Triterpene compositions and methods for use thereof

ABSTRACT

The invention provides novel saponin mixtures and compounds which are isolated from the species  Acacia victoriae  and methods for their use. These compounds may contain a triterpene moiety, such as acacic or oleanolic acid, to which oligosaccharides and monoterpenoid moieties are attached. The mixtures and compounds have properties related to the regulation of apoptosis and cytotoxicity of cells and exhibit potent anti-tumor effects against a variety of tumor cells.

BACKGROUND OF THE INVENTION

The present application is a continuation-in-part of co-pending U.S.Patent Application Ser. No. 60/099,066, filed Sep. 3, 1998, and acontinuation-in-part of U.S. Patent Application Ser. No. 60/085,997,filed May 19, 1998. The entire text of each of the above-referenceddisclosures is specifically incorporated by reference herein withoutdisclaimer.

FIELD OF THE INVENTION

The present invention relates generally to the field of medicine. Morespecifically, the invention relates to methods of obtaining novel plantcompounds having therapeutic uses in mammals.

DESCRIPTION OF RELATED ART

Plants are valuable sources for the identification of novel biologicallyactive molecules. One diverse class of molecules which has beenidentified in plants is the class of saponins. Saponins are highmolecular weight compounds comprising glycosides with a sugar moietylinked to a triterpene or steroid aglycone. Triterpene saponinsparticularly have been the subject of much interest because of theirbiological properties.

Pharmacological and biological properties of triterpene saponins fromdifferent plant species have been studied, including fungicidal,anti-viral, anti-mutagenic, spermicidal or contraceptive,cardiovascular, and anti-inflammatory activities (Hostettmann et al.,1995). Saponins are known to form complexes with cholesterol by bindingplasma lipids, thereby altering cholesterol metabolism (Oakenfull etal., 1983). Triterpene glycosides given in feed also have been shown todecrease the amount of cholesterol in the blood and tissues ofexperimental animals (Cheeke, 1971). Saponins have been found to beconstituents of many folk medicine remedies and some of the morerecently developed plant drugs.

The triterpene glycyrrhetinic acid, and certain derivatives thereof, areknown to have anti-ulcer, anti-inflammatory, anti-allergic,anti-hepatitis and antiviral actions. For instance, certainglycyrrhetinic acid derivatives can prevent or heal gastric ulcers (Dollet al., 1962). Among such compounds known in the art are carbenoxolone(U.S. Pat. No. 3,070,623), glycyrrhetinic acid ester derivatives havingsubstituents at the 3′ position (U.S. Pat. No. 3,070,624), amino acidsalts of glycyrrhetinic acid (Japanese Patent PublicationJP-A-44-32798), amide derivatives of glycyrrhetinic acid (Belgian PatentNo. 753773), and amide derivatives of 11-deoxoglycyrrhetinic acid(British Patent No. 1346871). Glycyrrhetinic acid has been shown toinhibit enzymes involved in leukotriene biosynthesis, including5-lipoxygenase activity, and this is thought to be responsible for thereported anti-inflammatory activity (Inoue et al., 1986).

Betulinic acid, a pentacyclic triterpene, is reported to be a selectiveinhibitor of human melanoma tumor growth in nude mouse xenograft modelsand was shown to cause cytotoxicity by inducing apoptosis (Pisha et al.,1995). A triterpene saponin from a Chinese medicinal plant in theCucurbitaceae family has demonstrated anti-tumor activity (Kong et al.,1993). Monoglycosides of triterpenes have been shown to exhibit potentand selective cytotoxicity against MOLT-4 human leukemia cells (Kasiwadaet al., 1992) and certain triterpene glycosides of the Iridaceae familyinhibited the growth of tumors and increased the life span of miceimplanted with Ehrlich ascites carcinoma (Nagamoto et al., 1988). Asaponin preparation from the plant Dolichos falcatus, which belongs tothe Leguminosae family, has been reported to be effective againstsarcoma-37 cells in vitro and in vivo (Huang et al., 1982). Soyasaponin, also from the Leguminosae family, has been shown to beeffective against a number of tumors (Tomas-Barbaren et al., 1988).Oleanolic acid and gypsogenin glycosides exhibiting haemolytic andmolluscicidal activity have been isolated from the ground fruit pods ofSwartzia madagascariensis (Leguminosae) (Borel and Hostettmann, 1987).

Genistein, a naturally occurring isoflavonoid isolated from soyproducts, is a tyrosine kinase inhibitor that has been shown to inhibitthe proliferation of estrogen-positive and estrogen-negative breastcancer cell lines (Akiyama et al., 1987). Inositol hexaphosphate (phyticacid), which is abundant in the plant kingdom and is a natural dietaryingredient of cereals and legumes, has been shown to cause terminaldifferentiation of a colon carcinoma cell line. Phytic acid alsoexhibits anti-tumor activity against experimental colon and mammarycarcinogenesis in vivo (Yang et al., 1995). Some triterpene aglyconesalso have been demonstrated to have cytotoxic or cytostatic properties,i.e., stem bark from the plant Crossopteryx febrifuga (Rubiaceae) wasshown to be cytostatic against Co-115 human colon carcinoma cell line inthe ng/ml range (Tomas-Barbaren et al., 1988).

While the previous reports have identified triterpene compounds whichhave any of a number of uses, there still is a great need in the art forthe identification of novel biologically active triterpene compounds.Many of these compounds are toxic to normal mammalian cells. Stillfurther, the biological activities of previously identified triterpenesvary widely and many posses limited or varying degrees of efficacy inthe treatment of any given human or mammalian condition. The greatdiversity of different triterpenes which have been identified and thegreat range of differences and unpredictability in the biologicalactivities observed among even closely related triterpene compounds,underscores the difficulties which have been encountered in obtainingtriterpenes which are potential therapeutic agents. Achieving thedifficult goal of identifying novel triterpenes with beneficialbiological activities could provide entirely new avenues of treatmentfor a diverse set of human ailments in which therapeutic optionscurrently are limited.

SUMMARY OF THE INVENTION

The present invention relates to the novel use of Acacia victoriae(Benth.) (Leguminosae) pods and roots for the isolation of novelbiologically useful compounds. Acacia victoriae seeds have been used asa source of food material by the indigenous people of Australia forgenerations (Lister et al., 1996). However, the pods and roots werediscarded as waste material. Therefore, the inventors of the presentinvention have demonstrated the presence of novel anti-cancer and otherbiologically useful compounds from the parts of the plant that were notused before. For example, the novel biologically active saponincompounds disclosed herein are often specifically cytotoxic to malignantcells.

In one embodiment the present invention provides novel saponin compoundsand mixtures thereof which may be isolated from the species Acaciavictoriae and methods for their use. In this respect, one embodiment ofthe invention provides a saponin composition comprising a triterpene orother aromatic terpenoid composition. The saponins disclosed herein mayalso contain a glycosidic group.

For preferred embodiments where the saponin contains a triterpenemoiety, this triterpene moiety is typically an acacic or oleanolic acidor other structurally similar triterpenoid moiety. The triterpene ortriterpene glycoside compositions may also typically comprise amonoterpene moiety or moieties and one of skill in the art willappreciate that the saponin compositions described herein may be furthersubstituted with other chemical functionalities. Thus, the saponincompounds disclosed herein may comprise a triterpene moiety attached toat least one, and preferably two, three, or more, monoterpene moieties.When more than one monoterpene moiety is present, these moieties mayeach be attached (i) directly to the triterpene moiety, (ii) to a sugar,or other linking group, which is attached to the triterpene moiety, or(iii) to a monoterpene moiety which is attached to the triterpene moietydirectly or through a sugar or other linking groups. Linking groupsinclude sugars, acyl, amide, alkoxy, ketyl, alkyl, alkylene and othersimilar chemical moieties which would be apparent to one of skill in theart. The triterpene glycosides disclosed herein typically have amolecular weight in the range of 1800 to 2600 amu, or from at least1800, 1900, 2000, 2100 amu to about 2200, 2300, 2400 or 2600 amu.

An important aspect of the invention provides the isolation of a mixturecomprising one or more isolated saponins or triterpene glycosides thatmay be characterized by the following properties: a) isolatable from thetissues of Acacia victoriae; b) containing at least one triterpeneglycoside having a molecular weight of from about 1800 to about 2600amu; c) the ability to induce cytotoxicity in a Jurkat cell; and d) theability to induce apoptosis in a Jurkat cell.

In particular embodiments of the invention, the triterpene compositionmay be characterized by the following properties: ability to inducecytotoxicity in a Jurkat cell with an IC₅₀ of from about 0.12 to about0.40 μg/ml. In other embodiments of the invention, the apoptosis isinduced when administered to a Jurkat cell at a concentration of fromabout 100 to about 400 ng/ml. In further embodiments of the invention,the apoptosis is induced when administered to a Jurkat cell at aconcentration of from about 200 to about 250, 300, 350 or 400 ng/ml orfrom about 300 to about 350 or 400 ng/ml.

In still other embodiments of the invention, the apoptosis is measuredby the reorganization of plasma membrane of a Jurkat cell by annexinbinding. This may be measured by flow cytometry and the apotosis inducedmay be from 16-18%.

Another embodiments of the invention encompasses a mixture comprisingone or more isolated triterpene glycosides characterized by thefollowing properties: a) isolatable from the tissues of Acaciavictoriae, b) containing at least one triterpene glycoside having amolecular weight of from about 1800 to about 2600; and c) the ability toinduce the release of cytochrome c from mitochondria in a Jurkat cell.

Still other embodiments of the invention encompasses a mixturecomprising one or more isolated triterpene glycosides characterized bythe following properties: a) isolatable from the tissues of Acaciavictoriae; b) containing at least one triterpene glycoside having amolecular weight of from about 1800 to about 2600; and c) the ability toactivate caspase-3 in a Jurkat cell. wherein the Caspase activity is inthe range of from about 0.3 to about 1.6 fluorescence units/minutes/mg.

In still other embodiments of the invention, the mixture comprising oneor more isolated triterpene glycosides may be characterized by thefollowing properties: a) isolatable from the tissues of Acaciavictoriae; b) containing at least one triterpene glycoside having amolecular weight of from about 1800 to about 2600; and c) the ability tocause the cleavage of PARP in a Jurkat cell.

In further embodiments of the invention, the mixture comprising one ormore isolated triterpene glycosides may be characterized by thefollowing properties: a) isolatable from the tissues of Acaciavictoriae; b) containing at least one triterpene glycoside having amolecular weight of from about 1800 to about 2600 amu; and c) theability to inhibit the activity of PI-3-kinase in a Jurkat cell.

In yet other embodiments of the invention, the mixture comprising one ormore isolated triterpene glycosides may be characterized by thefollowing properties: a) isolatable from the tissues of Acaciavictoriae; and b) the ability to inhibit the initiation and promotion ofmammalian epithelial cells to a premalignant or malignant state.

In still other embodiments of the invention, the mixture comprising oneor more isolated triterpene glycosides may be characterized by thefollowing properties: a) isolatable from the tissues of Acaciavictoriae; and b) the ability to induce apoptosis in malignant mammaliancells.

An important aspect of the invention provides a nutraceuticalcomposition comprising a triterpene glycoside composition in apharmacologically acceptable medium such as a buffer, a solvent, adiluent, an inert carrier, an oil, a creme, or an edible material. Inone embodiment of the invention, the nutraceutical composition maycomprise dried and ground Acacia victoriae root, pod or combinationthereof in a pharmacologically acceptable medium. The nutraceuticalcompositions disclosed herein may typically be in the form of a tablet,a capsule, or an ointment.

In another aspect, the invention provides a process for preparing acomposition comprising a mixture of one or more isolated triterpeneglycosides, comprising: a) obtaining tissue from an Acacia victoriaeplant; b) extracting the tissue with a solvent to provide an extract;and c) obtaining one or more triterpene glycosides from the extract. Thetissues used in this process typically comprises pods, roots, seedlings,or mixtures thereof The solvent used for the extraction may be anyorganic solvent which is capable of extracting, often by dissolving, thesaponin compound of interest. Useful extraction solvents are methanol,ethanol, isopropyl alcohol, dichloromethane, chloroform, ethyl acetate,water, glycerol and mixtures thereof.

This process may include additional steps. For example, the process mayfurther comprise isolating the composition from plant bagasse byfiltration after the extracting. In a further embodiment, the processfurther includes the step of defatting the plant tissue with an organicsolvent prior to extracting. The organic solvent may be any solventsuitable for defatting, such as hexane, dichloromethane, chloroform,ethyl acetate or mixtures thereof. In another embodiment, the process ofisolation further comprises evaporating the solvent after theextracting.

This process may also comprise obtaining the mixture of the triterpenecompositions by chromatographically isolating at least triterpeneglycoside composition. Exemplary chromatographic techniques includeliquid chromatography, MPLC, or HPLC. Although solvents which may beemployed for the chromatographic isolation would be apparent to one ofskill in the art, exemplary solvents include methanol, acetonitrile,water, and mixture.

In yet another aspect, the invention provides a process for preparing acomposition comprising a mixture of one or more isolated triterpeneglycosides, comprising: a) preparing a tissue culture comprising cellsof an Acacia victoriae plant; and b) extracting the triterpenecomposition from the cells with a solvent thereby extracting at least afirst triterpene compound from the tissue. In one aspect, the tissueculture comprises a hairy root culture. In another aspect of theinvention, the tissue culture is prepared by infecting the cells ofAcacia victoriae with Agrobacterium rhizogenes R-1000. In a relatedaspect of the invention, the tissue culture comprises medium containingsucrose from about 3% to about 4% by weight. In another aspect of theinvention, the solvent used to extract the composition is methanol,ethanol, isopropyl alcohol, dichloromethane, chloroform, ethyl acetate,water or a mixture thereof.

In another aspect of the invention, the method further comprisesadditional steps, such as filtering plant bagasse from the triterpenemixture composition, isolating the triterpene mixture composition byliquid chromatography, and/or evaporating the solvent after theextracting step.

One aspect describes a method of continually propagating the tissues ofan Acacia victoriae plant from which one may extract the activecompounds of the invention. In one embodiment of the invention, a hairyroot tissue culture comprising cells of an Acacia victoriae plant whichhave been infected Agrobacterium rhizogenes R-1000 in a cell culturemedium is described. In a related embodiment, the tissue culture mediumcomprises sucrose from about 3% to about 4%.

Another aspect of the invention describes a method of continuallyharvesting an Acacia victoriae plant tissue comprising: a) cultivatingan Acacia victoriae plant in a hydroponic growth system; and b)harvesting tissue from the plant about 1 to about 4 times per year,wherein the harvesting does not kill the plant. In a related embodimentof the invention, the growth system is an aeroponic system. In anotherrelated embodiment of the invention, the tissue used for culture isAcacia victoriae root tissue.

An important aspect of this invention is a method of inhibiting theinitiation and promotion of mammalian epithelial cells to a premalignantor malignant state comprising administering to a the mammalian cell atherapeutically effective amount of the nutraceutical compositionsdescribed above. In one embodiment, the epithelial cell is a skin cell,a colon cell, a uterine cell, an ovarian cell, a pancreatic cell, aprostate cell, a renal cell, a lung cell, a bladder cell or a breastcell. In a related embodiment, the mammal is a human. In yet anotherrelated embodiment, the mode of administering the nutraceutical is oral.In yet another related embodiment of the invention, the mode ofadministering the nutraceutical is topical.

The invention also encompasses a method of inducing apoptosis in amalignant mammalian cell, comprising administering to the cell atherapeutically effective amount of a nutraceutical compositiondescribed above. In one embodiment, the cell is a skin cell, a coloncell, a uterine cell, an ovarian cell, a pancreatic cell, a prostatecell, a renal cell, a lung cell, a bladder cell or a breast cell. In arelated embodiment, the mammal is a human. In yet another relatedembodiment, the mode of administering the nutraceutical is oral. In analternative embodiment, the mode of administering the nutraceutical istopical.

The invention also encompasses a method of preventing the abnormalproliferation of mammalian epithelial cells in vitro or in a mammalcomprising administering to the mammalian cell or mammal atherapeutically effective amount of the nutraceutical compositionsdescribed above. In one aspect of the invention, the epithelial cellsare crypt cells. In another aspect of the invention the epithelial cellsare colon cells. In a related embodiment of the invention, the mammal isa human. In yet another related embodiment of the invention, the mode ofadministering the nutraceutical for in vivo application is oral.

The invention also contemplates a method of treating a mammal forinflammation, comprising administering to the mammal a therapeuticallyeffective amount of the nutraceutical compositions described above. In arelated embodiment of the invention, the mammal is a human.

The invention also comprises a purified triterpene compound comprising atriterpene moiety attached to a monoterpene moiety having the molecularformula:

or a pharmaceutical formulation thereof, wherein a) R₁ and R₂ areselected from the group consisting of hydrogen, C1-C5 alkyl, and anoligosaccharide; b) R₃ is selected from the group consisting ofhydrogen, hydroxyl, C1-C5 alkyl, C1-C5 alkylene, C1-C5 alkyl carbonyl, asugar, and a monoterpene group; and c) the formula further comprises R₄,wherein R₄ is selected from the group consisting of hydrogen, hydroxyl,C1-C5 alkyl, C1-C5 alkylene, C1-C5 alkyl carbonyl, a sugar, C1-C5 alkylester, and a monoterpene group, and wherein R₄ may be attached to thetriterpene moiety or the monoterpene moiety. The invention alsocontemplates the compound wherein R₃ is a sugar. In related embodimentsof the invention, the sugar is selected from the group consisting ofglucose, fucose, rhamnose, arabinose, xylose, quinovose, maltose,glucuronic acid, ribose, N-acetyl glucosamine, and galactose. In otherrelated embodiments of the invention, the compound further comprises amonoterpene moiety attached to the sugar. The invention also comprises acomposition wherein R₃ has the following formula

wherein R5 is selected from the group consisting of hydrogen, hydroxyl,C1-C5 alkyl, C1-C5 alkylene, C1-C5 alkyl carbonyl, a sugar, C1-C5 alkylester, and a monoterpene group.

In one embodiment of the invention, R₅ is a hydrogen or a hydroxyl. Inanother embodiment of the invention, R₁ and R₂ each comprise anoligosaccharide. In still other embodiments of the invention R₁ and R₂each comprise a monosaccharide, a disaccharide, a trisaccharide or atetrasaccharide. In related embodiments of the invention R₁ and R₂ eachcomprise an oligosaccharide comprising sugars which are separately andindependently selected from the group consisting of glucose, fucose,rhamnose, arabinose, xylose, quinovose, maltose, glucuronic acid,ribose, N-acetyl glucosamine, and galactose. In further aspects of theinvention, at least one sugar is methylated.

In one embodiment of the invention, R₄ is attached to the triterpenemoiety through one of the methylene carbons attached to the triterpenemoiety. In another embodiment of the invention, the triterpene moiety isoleanolic acid instead of acacic acid.

Another embodiment of the invention describes a composition comprising atriterpene glycoside having the molecular formula:

or a pharmaceutical formulation thereof, wherein a) R₁ is anoligosaccharide comprising N-acetyl glucosamine, fucose and xylose; andb) R₂ is an oligosaccharide comprising glucose, arabinose and rhamnose.In a related embodiment the compound having the molecular formula:

or a pharmaceutical formulation thereof is described.

Another aspect of the invention describes the purification of acomposition comprising a triterpene glycoside having the molecularformula:

or a pharmaceutical formulation thereof wherein, a) R₁ is anoligosaccharide comprising N-acetyl glucosamine, fucose and xylose; andb) R₂ is an oligosaccharide comprising glucose, arabinose and rhamnose.A related aspect of the invention describes the purification andcharacterization of a composition having the molecular formula:

or a pharmaceutical formulation thereof.

Yet another aspect of the invention describes the purification of acomposition comprising a triterpene glycoside having the molecularformula:

or a pharmaceutical formulation thereof, wherein, a) R₁ is anoligosaccharide comprising N-acetyl glucosamine, glucose, fucose andxylose; and b) R₂ is an oligosaccharide comprising glucose, arabinoseand rhamnose. A related aspect of the invention, describes thepurification and characterization of a composition comprising having themolecular formula:

Another aspect of the invention relates to a composition comprising atriterpene moiety, an oligosaccharide and three monoterpene units. Inone embodiment the triterpene moiety is acacic acid or oleanolic acid.

An important aspect of the invention contemplates pharmaceuticalpreparations of the compounds purified and characterized. In oneembodiment the pharmaceutical preparation is in a pharmacologicallyacceptable medium comprising a buffer, a solvent, a diluent, an inertcarrier, an oil, a creme, or an edible material. In some aspects of theinvention, the pharmaceutical composition is contemplated to furthercomprises a targeting agent. In related aspects of the invention, thetargeting agent can direct the delivery of the pharmaceuticalcomposition to an epithelial cell. In a related embodiment of theinvention, the targeting agent comprises an antibody which binds to theepithelial cell.

In certain embodiments of the invention, the pharmaceutical compositioncomprises at least a second composition that can kill an epithelialcell.

The compounds of this invention have shown chemoprotective effects inmice exposed to the carcinogen DMBA. The invention therefore provides amethod of inhibiting the initiation and promotion of a mammalianepithelial cell to a premalignant or malignant state in a mammalcomprising administering to the mammal a therapeutically effectiveamount of the pharmaceutical compositions described above. In oneembodiment of the invention, the epithelial cell is a skin cell, a coloncell, a uterine cell, an ovarian cell, a pancreatic cell, a lung cell, abladder cell, a prostate cell, a renal cell, or a breast cell. In arelated embodiment of the invention, the mammal is a human. In yetanother related embodiment of the invention, the mode of administeringthe pharmaceutical is oral. In still another alternative embodiment ofthe invention, the mode of administering the pharmaceutical is topical.In still other alternative embodiment of the invention, the mode ofadministering the pharmaceutical is by intratumoral injection. In stillanother alternative embodiment of the invention, the mode ofadministering the pharmaceutical is intravenous. In still furtheralternative embodiments of the invention, the mode of administering thepharmaceutical comprises inhaling an aerosol.

The invention also contemplates the use of the pharmaceuticalpreparations of the invention in combination with other therapies. Inone embodiment the other therapy comprises irradiating the epithelialcell with X-ray radiation, UV-radiation, γ-radiation, or microwaveradiation.

The invention also envisions a method of inducing apoptosis in amalignant mammalian cell in a mammal comprising administering to themammal a therapeutically effective amount of the pharmaceuticalcompositions described herein. In one embodiment of the invention, thecell is a skin cell, a colon cell, a uterine cell, an ovarian cell, apancreatic cell, a lung cell, a bladder cell, a prostate cell, a renalcell, or a breast cell.

In one important aspect the invention provides a method of preventingthe abnormal proliferation of a mammalian epithelial cell in a mammalcomprising administering to the mammal a therapeutically effectiveamount of the pharmaceutical compositions described above. In oneembodiment the epithelial cell is a crypt cell. In another embodiment ofthe invention, the epithelial cell is a colon cell. In a relatedembodiment of the invention, the mammal is a human. In yet anotherrelated embodiment of the invention, the mode of administering thepharmaceutical is oral. In still another alternative embodiment of theinvention, the mode of administering the pharmaceutical is topical. Instill other alternative embodiment of the invention, the mode ofadministering the pharmaceutical is by intratumoral injection. In stillanother alternative embodiment of the invention, the mode ofadministering the pharmaceutical is intravenous. In still furtheralternative embodiments of the invention, the mode of administering thepharmaceutical comprises inhaling an aerosol. The invention alsocontemplates the use of the pharmaceutical preparations of the inventionin combination with other therapies. In one embodiment the other therapycomprises irradiating the epithelial cell with X-ray radiation,UV-radiation, γ-radiation, or microwave radiation.

The invention also contemplates a method of treating a mammal forinflammation comprising administering to the mammal a therapeuticallyeffective amount of the pharmaceutical compositions of the triterpenecompounds described herein. In a related embodiment of the invention,the mammal is a human. In yet another related embodiment of theinvention, the mode of administering the pharmaceutical is oral. Instill another alternative embodiment of the invention, the mode ofadministering the pharmaceutical is topical. In still furtheralternative embodiments of the invention, the mode of administering thepharmaceutical comprises inhaling an aerosol.

Another important aspect of this invention is a method of regulatingangiogenesis in a mammal comprising administering to the mammal atherapeutically effective amount of the pharmaceutical compositionsdescribed. The method may be when the mammal is a human.

Although several of the methods describe herein are in vivo methods itis contemplated that in vivo the triterpene glycoside compounds willexhibit similar effects.

In addition to providing methods of preventing or treating cancer withthe compounds of the invention, the inventors have provided a number ofother uses for the compounds of the invention. In particular, thecompounds of the invention may be used as solvents, antioxidants,anti-fungal and anti-viral agents, piscicides or molluscicides,contraceptives, antihelmintics, angiogenesis regulators, UV-protectants,expectorants, diuretics, anti-inflammatory agents, regulators ofcholesterol metabolism, cardiovascular effectors, anti-ulcer agents,analgesics, sedatives, immunomodulators, antipyretics, as agents fordecreasing capillary fragility, as agents to combat the effects ofaging, as agents for increasing skin collagen, as agents for enhancingpenile function and as agents for improving cognition and memory.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1: Effect of UA-BRF-004-DELEP-F001 on human tumor cell lines. FIG.1 demonstrates the growth inhibition exhibited by ovarian (SK-OV-3, HEY,OVCAR-3), breast (MDA-468), melanoma (A375-M, Hs294t) and humanepidermoid (A431) cell lines treated with a crude legume plant extract.

FIG. 2: Effect of UA-BRF-004-DELEP-F023 (Fraction 23) on transformed andnontransformed cell lines. FIG. 2 demonstrates the cytotoxicityexhibited by fraction 23 on ovarian (SK-OV-3, OCC1, HEY, OVCAR-3),T-cell leukemia (Jurkat), prostate (LNCaP), fresh human ovarian tumorcells (FTC), human fibroblast (FS) and endothelial (HUVEC) cells. Only15-17% cytotoxicity was observed on nontransformed cells compared to the50-95% cytotoxicity shown by tumor cells.

FIG. 3: Effect of Fraction 35 (“UA-BRF-004-DELEP-F035” or “F035”) onhuman tumor cell lines. FIG. 3 demonstrates the cytotoxicity exhibitedby Fraction 35 treated human ovarian (HEY, OVCAR-3,C-1, SK-OV-3),pancreatic (PANC-1) and renal (769-P,786-O,A498) cell lines. The IC₅₀for the cell lines ranged from 1-6 μg/ml.

FIG. 4: Effect of Fraction 35 on Leukemia cell lines. FIG. 4 shows thatFraction 35 exhibited potent cytotoxicity against Jurkat (T-cellleukemia) cells with an IC₅₀ of 130 ng/ml and IC₅₀ for REH, KG-1 andNALM-6 (B-cell leukemia) cells in the range of 1-3 μg/ml.

FIG. 5: Effect of Fraction 35 on endothelial cell proliferation. FIG. 5shows that Fraction 35 is a potent inhibitor of endothelial cellproliferation with or without stimulation with bFGF.

FIG. 6: Effect of Fraction 35 on migration of capillary endothelialcells. FIG. 6 shows no effect on the migration of capillary endothelialcells suggesting lack of toxicity.

FIG. 7: Shows thin layer chromatography of,seedling and callus extracts.Lane 1, stem callus developed on BA-IAA medium; Lane 2, root callusdeveloped on BA-IAA medium; Lane 3, hypocotyl callus; Lane 4, seedlingstreated with methyl jasmonate (100 μM) on semi-solid medium; Lane 5,seedling control growing on semi-solid medium; Lane 6, standard F023;Lane 7, shoot developed on BA medium; Lane 8, seedling treated with 50μM methyl jasmonate; Lane 9, seedling treated with 100 μM methyljasmonate; Lane 10, seedling treated with 200 μM methyl jasmonate; Lane11, seedling control; and Lane 12, standard F023.

FIG. 8: Shows a photograph of the SENCAR mouse on the left and a crossof SENCAR and C57B1 on the right. Both were treated with repetitive 100nmol DMBA doses for 8 weeks. At 15 weeks both had numerous papillomasbut the cross of SENCAR and C57B1 mouse had fewer and smallerpapillomas. The C57B1 strain is resistant to carcinogenesis and will notdevelop tumors.

FIGS. 9A-F: Show epidermal sections of mice treated with acetone, DMBAor DMBA+UA-BRF-004-DELEP-F035. FIG. 9A: acetone treatment at 4 weeks.FIG. 9B: acetone treatment at 8 weeks. FIG. 9C: DMBA treatment at 4weeks. FIG. 9D: DMBA treatment at 8 weeks. FIG. 9E:DMBA+UA-BRF-004-DELEP-F035 treatment at 4 weeks. FIG. 9F:DMBA+UA-BRF-004-DELEP-F035 treatment at 8 weeks.

FIGS. 10A,B: Show the antioxidant effect on DNA of UA-BRF-004-DELEP-F035after 4 weeks. FIG. 10A: shows the antioxidant effects followingtreatment with a low concentration of UA-BRF-004-DELEP-F035 (0.1 mg/0.2ml). FIG. 10B: shows the antioxidant effects following treatment with ahigh concentration of UA-BRF-004-DELEP-F035 (0.3 mg/0.2 ml).

FIGS. 11A,B: Show the epidermal thickness after 4 weeks of treatmentwith DMBA and UA-BRF-004-DELEP-F035. FIG. 11A: shows the effect onepidermal thickness following treatment with a low concentration ofUA-BRF-004-DELEP-F035 (0.1 mg/0.2 ml). FIG. 11B: shows the effect onepidermal thickness following treatment with a high concentration ofUA-BRF-004-DELEP-F035 (0.3 mg/0.2 ml).

FIG. 12: Shows the percent increase in epidermal thickness after 4 weeksfollowing treatment with DMBA at low (0.1 mg/0.2 ml) or high (0.3 mg/0.2ml) concentration of UA-BRF-004-DELEP-F035.

FIG. 13: Shows the percent reduction in papillomas after 8 weeksfollowing treatment with DMBA at a low (0.1 mg/0.2 ml) or high (0.3mg/0.2 ml) concentration of UA-BRF-004-DELEP-F035.

FIG. 14: Shows an autoradiograph of a PCR reaction showing amplificationof mouse H-ras codon 61 mutation.

FIG. 15: Shows the initial strategy employed for purifying and isolatingthe biologically active triterpene compounds from Acacia victoriae.

FIG. 16: Shows a general, improved scheme for the purification,isolation, and characterization of the active constituents from Acaciavictoriae.

FIGS. 17A,B: FIG. 17A: shows an HPLC spectrum of acetylated sugarsisolated from the hydrolyzed active constituents found in Fraction 94(“UA-BRF-004Pod-DELEP-F094” or F094). FIG. 17B: shows an HPLC spectrumof acetylated sugars isolated from the hydrolyzed active constituentsfound in F094.

FIGS. 18A-F: FIG. 18A: shows an HPLC spectra of UA-BRF-004-DELEP-F035and F035-B2. FIG. 18B: shows an HPLC spectra ofUA-BRF-004Pod-DELEP-F094. FIG. 18C: shows an HPLC spectra of F140. FIG.18D: shows an HPLC spectra of F142. FIG. 18E: shows an HPLC spectra ofF144. FIG. 18F: shows an HPLC spectra of F145.

FIGS. 19A,B: Cell cycle analysis of OVCAR-3 cells pre and post treatment(48 h) with Fraction 35. The FIG. demonstrates that there is a ˜8%increase in the number of cells in G1 phase and 10% decrease of cells inS phase of cell cycle post treatment with Fraction 35 showing a G1arrest. FIG. 19A: cell cycle analysis of untreated OVCAR-3 tumor cells.FIG. 19B: cell cycle analysis of OVCAR-3 tumor cells treated withFraction 35.

FIG. 20: EMSA demonstrating marked inhibition of TNF activated NF-κB byexposure of cells to UA-BRF-004-DELEP-F035 and UA-BRF-004Pod-DELEP-F094.Treatments were as follows: lane 1, untreated; lane 2, TNF (100 pM);lane 3, UA-BRF-004-DELEP-F035 (1 μg/ml); lane 4, TNF+F035 (1 μg/ml);lane 5, F035 (2 μg/ml); lane 6, TNF+F035 (2 μg/ml); lane 7, F094 (1μg/ml); lane 8, TNF+F094 (1 μg/ml); lane 9, F094 (2 μg/ml); lane 10,TNF+F094 (2 μg/ml).

FIG. 21: Lipid kinase assay demonstrating inhibition of PI3-Kinase byUA-BRF-004-DELEP-F035 and wortmannin.

FIG. 22: SDS-PAGE gel analyzed by western-ECL using phospho-specific AKTand total AKT antibody. Post treatment of cells with 1 and 2 μg/ml ofUA-BRF-004-DELEP-F035 caused a marked inhibition of AKT phosphorylation(active AKT), which was similar to a 2 hour treatment of cells with 1 μMof wortmannin.

FIG. 23: Discloses PCR™ amplification of a portion of rol B gene fromfour independently transformed root clones. (Lanes, L-R, 1: Kb ladder,2: positive control (Plasmid DNA from R1000 strain), 3: negative control(DNA from non-transformed root). 4-7: four independently transformedroot clones: Note the amplification of a 645 by fragment in positivecontrol and transformed roots.

FIG. 24: Structure of Elliptoside A and Elliptoside E (Beutler, 1997).

FIG. 25: HPLC separation of the constituents in F094.

FIG. 26: HPLC separation of the constituents in F035.

FIG. 27: First-fractionation by semi-prep HPLC of F094.

FIG. 28: Second-fractionation by semi-prep HPLC of F094.

FIG. 29: Preparative -fractionation of F094.

FIG. 30: Analysis of preparative-fraction D.

FIG. 31: Analysis of preparative-fraction G/H.

FIG. 32: Compound G1 after second PFP column purification.

FIG. 33: Compound G1 after final C-18 purification.

FIG. 34: Compound D1 after Waters C-18 column purification.

FIG. 35: Compound D1 after final C-18-Aq purification.

FIG. 36: Depicts compounds from the degradation of compound D1.

FIG. 37: Depicts compounds from the degradation of compound G1.

FIG. 38: Depicts compounds from the degradation of compound B1.

FIG. 39: Structure of triterpene glycoside D1

FIG. 40: Structure of triterpene glycoside G1

FIG. 41: Structure of triterpene glycoside B1

FIG. 42: Effect of mixture of triterpene glycosides (F035) on cancer andnormal cell lines: F035 was evaluated for cytotoxicity by the proceduresdescribed in the examples. The activity of F035 was examined on panel ofcancer and normal cell lines as shown in the FIG. The IC₅₀ ranged from0.2-5.8 μg/ml for cancer cell lines. No significant cytotoxicity wasobserved (IC₅₀ 15 μg/ml to >25 μg/ml) on normal and immortalized celllines.

FIG. 43: Cytotoxicity profile of purified triterpene glycosides D1 andG1 on human cancer cell lines: The purified extracts were evaluated fortheir activity on following human cancer cell lines: Jurkat (T-cellleukemia), C-2 Hey Variant (ovarian), 769-P (renal), MDA-MB-231,MDA-MB-453 (breast). The results are shown as mean+SEM.

FIG. 44: Effect of purified compounds D1 and G1 and a mixture oftriterpene glycosides (F035) on apoptosis: Apoptosis was measured usingAnnexin V binding assay in which the cells were stained with annexinV-FITC and for DNA content with propidium iodide (PI) and analyzed usingflow cytometry. Cells were incubated for 16 hours with 0.5-1.0 μg/ml ofextracts. After 16 hours of treatment, three populations of cells wereobserved. Cells that had died or were in late stage of apoptosis(Annexin V-FITC and PI positive), cell undergoing apoptosis (AnnexinV-FITC positive and PI negative), and the cells that were viable and notundergoing apoptosis (Annexin V-FITC and PI negative; lower leftquadrant).

FIGS. 45A,B: Inhibition of PI3-kinase activity and AKT phosphorylation:The ability to phosphorylate phosphatidylinositol (PI) was measured forp85 protein immunoprecipitates from cellular lysates. Autoradiograms ofthe in vitro kinase assay separated on thin layer chromatography for p85immunoprecipitates using Jurkat cells. FIG. 45B: Inhibition of AKTphosphorylation on Ser-473 and Thr-308 with crude and pure triterpeneglycosides. Jurkat cells were incubated with crude (F035) and purifiedextracts of D1 and G1 for 16 hours at 37° C. The cell lysates wereresolved on 9% SDS-PAGE and analyzed by western blot-ECL analysis usinganti Ser-473, Thr-308 and total AKT antibodies as probes.

FIGS. 46A-D:Inhibition of TNF-induced NF-kB and induction of iNOS withtriterpene glycosides: Jurkat cells were exposed to differentconcentrations of F035 (1-4 μg/ml; FIG. 46A) and 2 μg/ml of pureextracts (D1 and G1; FIG. 46B) for 16 hours and NF-kB was activated with100 pM of TNF for 15 mins at 37° C. The DNA-protein complex wasseparated on 7.5% native polyacrylamide gels and the radioactive bandswere visualized and quantitated by Phospholmager. NOS were induced inU-937 (FIG. 46C) and Jurkat (FIG. 46D) as described in Methods. Cellularprotein was resolved on SDS-PAGE and analyzed using western blot-ECLusing anti-iNOS antibody.

FIG. 47: Effect of F035 and D1 on cleavage of PARP in Jurkat cells.

FIG. 48: Effect of z-vad fmk on F035 induced PARP cleavage in Jurkatcells.

FIG. 49: Effect of F035, F094, D1 and G1 on caspase activity in Jurkatcells.

FIG. 50: Effect of F035 on cytochrome release from Jurkat mitochondria.

DETAILED DESCRIPTION OF THE INVENTION

The present invention seeks to overcome limitations in the prior art byproviding novel biologically active triterpene glycoside compositions.In particular, the present inventors have identified and purifiedtriterpene compounds from Acacia victoriae. The identified compoundsexhibit potent anti-tumor activity at concentrations where there islittle or no cytotoxicity to normal human cells.

The triterpene compounds of the invention were identified from atargeted screening of 60 plant extracts from selected leguminous speciesnative to and and semi-arid regions. Of the initial screening, oneextract, designated UA-BRF-004-DELEP-F001 and isolated from Acaciavictoriae (Benth.) (Leguminosae), showed potent anti-tumor activityagainst a variety of human tumor cell lines. This extract wassubsequently further purified into various fractions. In two rounds ofpurification, an extract was identified which comprised the purifiedanti-tumor compounds. This extract was identified to contain purifiedtriterpene glycoside saponins. A procedure was subsequently developedfor the efficient isolation of the active compounds.

Further testing of the more purified extract further elucidated thebiological activities of the extract. The purified extract demonstratedenhanced anti-tumor activity relative to the crude extract, inconcentrations that exhibited little or no toxicity to normal humancells. The extract was still further shown to have a chemoprotectiveeffect in mice exposed to carcinogens.

The plant from which the extract was isolated, Acacia victoriae, wasselected based on factors including native environment and limited priorstudy of the species. Acacia victoriae originates from Australia, buthas been introduced as a horticultural variety throughout the world andis commonly known as prickly wattle or elegant wattle. The tree grows ata rate of 60 to 120 cm per year, is tardily drought deciduous and ishardy to at least −15° C. Mature plants grow to 10-15 feet and havebluish-green bipinnate leaves. In the southwest United States, the planttypically flowers from April to May, with pods ripening in June. Acaciavictoriae has a number of agricultural uses, including wind breaks,shelter belts, food, critical area stabilization, and as a low water-useornamental. Different Acacia species seeds have been used as a source offood material by the indigenous people of Australia for generations(Lister et al., 1996). Among the Acacia's, Acacia victoriae is the mostcommon and widespread species, present all over Australia, aretherefore, the most widely consumed species. Acacia seeds, commonlycalled wattleseed, are in high demand for use as a ground product inpastries and breads and also as a flavoring in desserts, especiallyice-cream. They are also used to produce a high quality coffee-likebeverage and among the Acacia species, Acacia victoriae (Benth.) isgenerally regarded as having a superior flavor (Lister et al., 1996).However, there is no record of the use of pods and roots of this plant.

The present invention relates to the novel use of Acacia victoriae podsand roots for the isolation of biologically useful compounds. Theinventors of the present invention have demonstrated the presence ofnovel anti-cancer and other biologically useful compounds from parts ofthe plant that were not used before.

II. Purification and Identification of the Triterpenes of the Invention

An important aspect in the use of plant extracts as pharmaceuticalpreparations is the characterization and determination of the individualactive constituents. Such also is the case for triterpene saponinpreparations, which often require sophisticated techniques for theisolation, structure elucidation and analysis of their components andglycosides. When biological testing of the pure compounds is to beperformed, it is necessary to isolate them in sufficient quantity andpurity.

Since triterpenes and other related saponins have relatively largemolecular weights and are of high polarity, their isolation can bechallenging. A problem involved in the isolation of pure saponins is thepresence of complex mixtures of closely related compounds, differingsubtly either in the nature of the aglycone or the sugar part (nature,number, positions and chirality of attachment of the monosaccharides).Difficulties also are encountered with labile substituents such asesters. For example, the major genuine soybean saponin, a γ-pyronederivative (BOA), is only extracted by aqueous ethanol at roomtemperature. Extraction with heating (80° C.) leads to fission of theester moiety and formation of soyasaponin I (Bb) (Kudou et al., 1992).In plants, saponins are accompanied by very polar substances, such assaccharides and coloring matter, including phenolic compounds and thelike, are not easily crystallized, and can be hygroscopic, making iteven more difficult to obtain crystals.

Characterization of pure saponins also is challenging because of thelack of crystalline material. Melting points are imprecise and oftenoccur with decomposition. Therefore, determinations of sample puritywill not generally be made only based on the melting point, opticalrotation value or another physical constant. A better test of the purityof a saponin can be obtained by TLC or HPLC examination—if possible byco-chromatography with an authentic sample. The coloration of spots onTLC plates after spraying with suitable reagents is an additionalindicator of potential individual components. For example, one of thetriterpene glycosides of the invention, D1, has a HPLC retention time of15.2 minutes. This is different from another related compound,elliptoside E, isolated from Archidendron ellipticum, by John Beutler etal., 1997, which has a HPLC retention time of 12.5 minutes. Furthercharacterization of the triterpenes of the invention show that thisdifference in retention time are at least due to differences inchirality and in the double bonds of D1 and the reported features ofelliptoside E.

(i) Chemical Purifications

Chemical purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofa plant extract into the triterpene glycoside compounds describedherein. Having generally separated the compounds of the invention fromplant material, the triterpene glycosides of interest may be furtherpurified using the techniques described herein, for example,chromatographic techniques, to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure triterpene glycoside composition arespecifically disclosed herein below.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of triterpeneglycosides from plant material. In a preferred embodiment of theinvention, the triterpene glycosides are purified from a plant of thefamily Leguminosae, or more preferably from the genus Acacia, and mostpreferably from the species Acacia victoriae and further more preferablyfrom the species Acacia victoriae (Bench.). The term “isolatedtriterpene glycoside” as used herein, is intended to refer to acomposition, isolatable from other components, wherein the compositionis purified to any degree relative to its naturally-obtainable state.

Generally, “isolated” will refer to an organic molecule or group ofsimilar molecules that have been subjected to fractionation to removevarious other components, and which composition substantially retainsits expressed biological activity. Where the term “substantiallypurified” is used, this designation will refer to a composition in whichtriterpene glycosides form the major component of the composition, suchas constituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the molecules in the composition.

There is no general requirement that the triterpene compositions of theinvention always be isolated and provided in their most purified state.Indeed, it is contemplated that less substantially purified productswill have utility in certain embodiments. For example, the inventorsenvision the use of dried Acacia victoriae root and pod and extractsthereof as nutraceuticals. Nutraceuticals by definition contain amixture of different bioactive compounds that synergistically havebeneficial effects on health. The nutraceuticals of the presentinvention may be in the form of tablets or capsules and can be takenorally or alternately may contain extracts of the plant in an ointmentwhich can be applied topically. Partial purification may be accomplishedby using fewer purification steps in combination, or by utilizingdifferent forms of the same general purification scheme. For example, itis appreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater “-fold”purification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of product, or inmaintaining the biological activity of the triterpene compounds.

(ii) Extraction and Preliminary Purification

Extraction procedures should be as mild as possible because certainsaponins can undergo transformations including enzymatic hydrolysisduring water extraction, esterification of acidic saponins duringalcohol treatment, hydrolysis of labile ester groups and transacylation.Therefore, care should be taken to follow the individual steps in anisolation procedure, for example, in thin layer chromatography.

Although numerous variations are possible, current general proceduresfor obtaining crude saponin mixtures typically include extraction withmethanol, ethanol, water or aqueous alcohol; a defatting step, generallywith petroleum ether, performed before the extraction step or on theextract itself; dissolution or suspension of the extract in water;shaking or washing the solution or suspension with n-butanol saturatedwith water; and precipitation (optional) of saponins with diethyl etheror acetone. A dialysis step also can be included in order to removesmall water-soluble molecules such as sugars (see, for example, Zhou etal., 1981; Massiot et al., 1988).

The most efficient extraction of dry plant material is achieved withmethanol or aqueous methanol. Methanol is also used for fresh plantmaterial. Although water is typically a less efficient extractionsolvent for saponins (unless specifically water-soluble glycosides aredesired) it has the advantages of being easily lyophilized and giving acleaner extract. Depending on the proportion of water used forextraction, either monodesmosidic or bidesmosidic saponins may beobtained (Domon and Hostettmann, 1984; Kawamura et al., 1988). Freshvegetable material contains active enzymes (esterases) which, whenhomogenized with a solvent, are able to convert bidesmosides intomono-desmosides. Even dry material may contain esterases which areactivated in the presence of water. In the case of momordin I (amonodesmosidic oleanolic acid saponin) it was found that conversion tomomordin II (the corresponding bidesmoside) takes place in water and in30% and 60% methanol solutions, but not in 80% and 100% methanolsolutions. On the contrary, homogenates of the fresh roots in methanolretained enzyme activity. However, the enzymes could be inactivated byfirst soaking the fresh roots in 4% hydrochloric acid and thebidesmoside was then shown to be the major component. It is, therefore,clear that the correct choice of extraction procedure is an extremelyimportant first step.

Methods typically used to purify proteins, such as dialysis,ion-exchange chromatography and size-exclusion chromatography, areuseful in partially separating saponins in aqueous solution fromnon-saponin components, but are generally ineffective in separatingindividual saponins because of the tendency of saponins to form mixedmicelles. Hence, effective separation typically requires the use oforganic solvents or solvent/water systems that solubilize theamphiphilic saponins as monomers so that the formation of mixed micellesdoes not interfere with separation.

A common problem observed for furostanol saponins is the formation of22-OCH₃ derivatives during extraction with methanol. However, thegenuine 22-hydroxyfurostanols can either be obtained by extraction withanother solvent (e.g., pyridine) or by treatment of the methoxylatedartifacts with boiling aqueous acetone (Konishi and Shoji, 1979).

(iii) Thin-Layer Chromatography (TLC)

The qualitative analysis of triterpene saponins by TLC is of greatimportance for all aspects of saponin investigations. TLC plates(usually silica gel) can handle both pure saponins and crude extracts,are inexpensive, rapid to use and require no specialized equipment. Anumber of visualization reagents are available for spraying onto theplates (Table 2). Methods of preparation of the most common reagents areas follows:

-   -   Vanillin-sulfuric acid (Godin reagent). A 1% solution of        vanillin in ethanol is mixed in a 1:1 ratio with a 3% solution        of perchloric acid in water and sprayed onto the TLC plate. This        is followed by a 10% solution of sulfuric acid in ethanol and        heating at 110° C.    -   Liebermann-Burchard reagent. Concentrated sulfuric acid (1 ml)        is mixed with acetic anhydride (20 ml) and chloroform (50 ml).        Heating at 85-90° C. gives the required coloration on the TLC        plate.    -   Antimony(III) chloride. The TLC plate is sprayed with a 10%        solution of antimony chloride in chloroform and heated to 100°        C.    -   Anisaldehyde-sulfuric acid. Anisaldehyde (0.5 ml) is mixed with        glacial acetic acid (10 ml), methanol (85 ml) and concentrated        sulfuric acid (5 ml). This solution is sprayed onto the TLC        plate, which is then heated at 100° C.

Spraying with vanillin-sulfuric acid in the presence of ethanol andperchloric acid, for example, gives a blue or violet coloration withtriterpene saponins. With anisaldehyde-sulfuric acid, a blue orviolet-blue coloration is produced on heating the TLC plate. SprayingTLC plates with a solution of cerium sulphate in sulfuric acid givesviolet-red, blue or green fluorescent zones under 365 nm UV light(Kitagawa et al., 1984b). In some cases, simply spraying the plates withwater is sufficient to reveal the saponins present. Additional sprayreagents may be found in, for example, Stahl (1969).

The most frequently used solvent for TLC is chloroform-methanol-water(65:35:10), but other solvents are also useful. The solventn-butanol-ethanol-ammonia (7:2:5) is especially useful for glycosidescontaining uronic acid residues; i.e., for very polar mixtures. Otherwidely used solvents include n-butanol-acetic acid-water (4:1:5; upperlayer) or chloroform-methanol-acetic acid-water (60:32:12:8).

Systems employed for the TLC of glycoalkaloids typically include ethylacetate-pyridine-water (30:10:30; upper phase). Visualization is withsteroid reagents (anisaldehyde-sulfuric acid) or with alkaloid reagents(Dragendorff reagent, cerium(IV) sulphate). Other TLC solvents andvisualization reagents are given by Jadhav et al. (1981) and BaerheimSvendsen and Verpoorte (1983).

Numerous quantitative determinations are possible with TLC. For example,the density of spots obtained with a suitable spray reagent can bemeasured directly using a densitometer. Alternatively, quantitativedeterminations are possible by carrying out TLC separations, scrapingthe relevant band off the plates (located, for example, with iodinevapor), eluting the saponin and measuring the UV absorbance afteraddition of a suitable reagent (e.g., concentrated sulfuric acid).

Reversed-phase TLC plates are commercially available and provide anexcellent analytical method for saponins which is complementary to TLCon silica gel plates. Almost exclusive use of methanol-water andacetonitrile-water mixtures is made for developing reversed-phase plates(for example, Merck RP-8 or RP-18 HPTLC plates). Alternatively, DIOLHPTLC glass-backed plates may be used. These can be used with normalsilica gel TLC-type solvents or with methanol-water andacetonitrile-water solvents, as for RP-TLC.

Exemplary reagents for TLC detection and for the spectrophotometric andcolorimetric determination of saponins are listed below, in Table 2.

1. Centrifugal Thin-Layer Chromatography (CTLC)

The CTLC technique is a planar method related to preparative thin-layerchromatography (TLC) but without the need to scrape bands off the TLCplate (Hostettmann et al., 1980). CTLC relies on the action of acentrifugal force to accelerate mobile phase flow across a circular TLCplate. The plate, coated with a suitable sorbent (1, 2 or 4 mmthickness), is rotated at approximately 800 r.p.m. by an electric motor,while sample introduction occurs at the center and eluent is pumpedacross the sorbent. Solvent elution produces concentric bands across theplate. These are spun off at the edges and collected for TLC analysis.Separations of 50-500 mg of a mixture on a 2 mm sorbent layer arepossible.

A combination of CTLC with chloroform--methanol-water (100:30:3) andcolumn chromatography has been described for the isolation ofginsenosides (Hostettmann et al., 1980). Saponins also have beenobtained with chloroform-methanol-water mixtures on silica gel plates.Two protoprimulagenin A glycosides from Eleutherococcus senticosus roots(Araliaceae) were purified by CTLC (chloroform-methanol-water 65:35:7)after column chromatography on silica gel and gel filtration on SephadexLH-20 (Segiet-Kujawa and Kaloga, 1991). For the isolation of cycloartaneglycosides from Passiflora quadrangularis (Passifloraceae), the solventsystem ethyl acetate-ethanol-water (8:2:1 or 16:3:2) was used at a flowrate of either 1 ml/min (Orsini et al., 1987) or 1.5 ml/min (Orsini andVerotta, 1985).

A Hitachi centrifugal liquid chromatograph, model CLC-5, has beendescribed for use in separation of saponins. Chromatography is carriedout with this machine on silica gel plates with the eluentchloroform-methanol-water (7:3:1 (lower phase)→65:35:10 (lower phase)).Using this technique a total of 1 g of semi-purified saponin fractionwas chromatographed on the circular plate (Kitagawa et al., 1988;Taniyama et al., 1988).

(iv) Open-Column Chromatography

A number of the classical solvent systems employed for the silica gelcolumn chromatography of saponins have previously been described and maybe found in, for example, Woitke et al., 1970 and Adler and Hiller,1985. Open-column chromatography is often used as a first fractionationstep for a crude saponin mixture, but in certain cases may yield pureproducts. In general, though, the resolution is not high and complexmixtures are only partially separated. Other problems are the loss ofmaterial because of irreversible adsorption and the length of timerequired to perform the separations.

Silica gel chromatography with chloroform-methanol-water eluents is oneof the most widely applicable techniques. When a biphasic system isused, the water-saturated chloroform phase is the eluent. Thus, agradient of chloroform-methanol-water (e.g., 65:35:5→65:40:10) can beemployed for the initial separation of a methanol extract of planttissue on silica gel. Further chromatography on low-pressure columns canbe used to yield, for example, a monodesmosidic molluscicidal saponin,while a bidesmosidic saponin can be obtained by silica gel columnchromatography with a solvent system such as acetone-n-propanol-water(35:35:5) (Borel et al., 1987).

A complex mixture of triterpene glycosides has been isolated from thecorms of Crocosmia crocosmiiflora (Iridaceae). Three of these,2,9,16-trihydroxypalmitic acid glycosides of polygalacic acid, wereobtained by a strategy involving open-column chromatography of a crudesaponin mixture on silica gel 60 (60-230 μm), employingn-butanol-ethanol-water (5:1:4, upper layer) andchloroform-methanol-water (60:29:6) as eluents. Final purification wasby HPLC (Asada et al., 1989).

Extensive use of silica gel chromatography has also enabled theseparation of the dammarane glycosides actinostemmosides A-D fromActinostemma lobatum (Cucurbitaceae). After an MCI (Mitsubishi ChemicalIndustries) polystyrene gel column, the relevant fractions werechromatographed with a variety of solvents: chloroform-methanol-water(7:3:0.5, 32:8:1), chloroform-methanol (9:1, 1,1), chloroform-ethanol(17:3), ethyl acetate-methanol (4:1), and chloroform-methanol-ethylacetate-water (3:3:4:1.5, lower layer). By this means, pureactinostemmoside C was obtained while actinostemmosides A and B requiredan additional low-pressure LC step and actinostemmoside D required afinal separation on a C-18 column eluted with 70% methanol (Iwamoto etal., 1987).

Certain ester saponins have been chromatographed on silica gelimpregnated with 2% boric acid (Srivastava and Kulshreshtha, 1986;1988).

As an addition to normal silica gel, coarse RP sorbents are now employedin the open-column chromatography of saponins. As long as thegranulometry is not too fine and the columns not too long, gravity-fedcolumns are quite suitable. RP chromatography is generally introducedafter an initial silica gel separation step and enables a change inselectivity for the substances being separated. Another possibility isto introduce the reversed-phase separation after a DCCC step (Higuchi etal., 1988).

1. Open-Column Chromatography with Polymeric Sorbents

The use of dextran supports, as found in Sephadex column packings, hasbeen current practice for a number of years. Sephadex LH-20 finds themost frequent application but the ‘G’ series of polymers is not withoutinterest.

In recent work on the isolation of saponins, a new generation ofpolymers has been exploited, particularly in Japan. Diaion HP-20(Mitsubishi Chemical Industries, Tokyo), for example, is a highly porouspolymer which is widely used for the initial purification steps.

Typically, the polymeric supports are washed with water after loadingthe sample in order to elute monosaccharides, small charged moleculessuch as amino acids, and other highly water-soluble substances. Elutionwith a methanol-water gradient (or with methanol alone) is thencommenced to obtain the saponin fractions. Other chromatographictechniques are employed for the isolation of pure saponins.

Elution of HP-20 gels with acetone-water mixtures has also beenreported. For example, in the isolation of bidesmosidic glycosides ofquillaic acid from the tuber of Thladiantha dubia (Cucurbitaceae),methanol extracts were passed through a column of Diaion CHP-20P andwashed with water. The crude saponins were eluted with 40% acetone.Further separation involved silica gel chromatography (ethylacetate-methanol-water 6:2:1) and HPLC (Nagao et al., 1990).

For the isolation of fibrinolytic saponins from the seeds of Luffacylindrica (Cucurbitaceae), a water extract was chromatographed on anAmberlite XAD-2 column eluted with methanol, followed by a second XAD-2column eluted with 40-70% methanol. The active principles were obtainedin the pure state after silica gel column chromatography withchloroform-methanol-water (65:35:10, lower layer→65:40:10) (Yoshikawa etal., 1991).

(v) Medium-Pressure Liquid Chromatography (MPLC)

When relatively large amounts of pure saponins are required, MPLC isvery useful. Unlike commercially available LPLC equipment, gramquantities of sample can be loaded onto the columns, while separationsare run at pressures of up to 40 bar. The granulometry of the supportnormally lies in the 25-40 μm range and separations are rapid, requiringconsiderably less time than open-column chromatography. A directtransposition of separation conditions from analytical HPLC to MPLC canbe achieved on reversed-phase supports, thus facilitating the choice ofsolvent (Hostettmann et al., 1986).

As an example, molluscicidal saponins from Cussonia spicata (Araliaceae)were obtained in sufficient quantities for biological testing by MPLC ona C-8 sorbent with methanol-water (2:1) (Gunzinger et al., 1986). Infact, this method required just two steps (one on a silica gel supportand the second on RP material) for isolation of saponins from a butanolextract of the stem bark.

The isolation of saponins also can be achieved by combination of MPLC,for example using a LiChroprep RP-8 (25-40 μm, 46×2.6 cm) column withmethanol-water mixtures in combination with rotation locularcountercurrent chromatography (RLCC) (Dorsaz and Hostettmann, 1986).Another MPLC technique uses axially compressed (Jobin-Yvon) columns(Elias et al., 1991).

Examples of support-solvent combinations which are useful in theseparation of triterpenes from plant extracts are given in Table 1,below.

TABLE 1 Applications of MPLC in the Separation of Triterpene SaponinsPlant Support Solvent Reference Cussonia spicata Silica CHCl₃—MeOH—H₂0Gunzinger gel (6:4:1) et al., 1986 C-8 MeOH—H₂0 (2:1) Gunzinger et al.,1986 Calendula C-8 MeOH—H₂0 (65:35, Chemli arvensis 73:27) et al., 1987C. officinalis Silica CHCl₃ MeOH H₂0 Vidal- gel (61:32:5) Ollivier etal., 1989 C-18 MeOH—H₂0 (60:40, Vidal- 80:20) Ollivier et al., 1989Polygala Silica CH₂Cl₂—MeOH H₂0 Hamburger chamaebuxus gel (80:20:2) andHostettmann, 1986 C-8 MeOH—H₂0 (55:45) Hamburger and Hostettmann, 1986Swartzia C-8 MeOH H₂0 (65:35) Borel and madagascariensis Hostettmann,1987 Talinum C-8 MeOH—H₂0 (60:40) Gafner tenuissimum et al., 1985Sesbania sesban C-8 MeOH—H₂0 (55:45, Dorsaz 60:40) et al., 1988Tetrapleura C-8 MeOH—H₂0 (70:30) Maillard tetraptera et al., 1989Albizzia lucida C-8 MeOH—H₂0 (6:4 → 9:1) Orsini et al., 1991 C-18MeOH—H₂0 (7:3) Orsini et al., 1991 Passiflora C-18 MeOH—H₂0 (17:3)Orsini and quadrangularis Verotta, 1985 Hedera helix C-18 MeOH—H₂0gradient Elias et al., 1991 Primula veris C-18 MeOH—H₂0 (5:5 → 7:3)Calis et al., 1992 Silica CHCl₃—MeOH—H₂0 Calis gel (61:32:7) et al.,1992 Steroid saponins Balanites Silica CHCl₃—MeOH—H₂0 Hosny aegyptiacagel (80:20:1 → 25:25:2 and et al., 1992 70:30:3)

(vi) High-Performance Liquid Chromatography (HPLC)

Chromatography by HPLC is a powerful technique for obtainingmulti-milligram quantities of saponins from mixtures of closely relatedcompounds and, in this respect, is very frequently employed as a finalpurification step. Whereas MPLC makes use of larger particles (25-100μm), semi-preparative HPLC sorbents lie in the 5-30 μm granulometryrange and consequently permit a higher separation efficiency.

Semi-preparative HPLC was employed to separate oleanolic acidtriglycoside from its partial hydrolysis products. This was necessary inorder to determine whether the galactose moiety was attached at positionC-3 or C-4 of the glucose residue. Isolation of isomeric saponins wasperformed on a 7 μm LiChrosorb RP-8 column (250×16 min) withacetonitrile-water (38:62) at a flow rate of 10 ml/min. Detection was at206 nm and from 50 mg of mixture (Décosterd et al., 1987).

A large-scale separation of saikosaponins a, c and d from Bupleurumfalcatum (Umbelliferae) roots has been achieved on axially compressedcolumns, dimensions 100×11 cm I.D. Preliminary purification of amethanol extract was carried out by solvent partition and chromatographyon HP-20 polymer. The preparative HPLC column was packed with C-18silica gel (20 μm particle size; 5 kg) and eluted at a flow rate of 210ml/min with an aqueous acetonitrile step gradient. A charge of 10 g wassufficient to give 400 mg of saikosaponin c, 1200 mg of saikosaponin aand 1600 mg of saikosaponin d (Sakuma and Motomura, 1987).

Ginsenosides have been isolated from Panax trifolius (Araliaceae) by atwo-step procedure, involving chromatography on a Waters Prep 500 system(radially compressed columns) with three silica gel cartridges (300×57min) arranged in series. The eluent was the upper phase ofn-butanol-ethyl acetate-water (4:1:5) and charges of 4 g were injected.Semi-preparative HPLC on a carbohydrate column (Waters, 300×7.8 mm) withacetonitrile-water (86:14 or 80:20) at a flow rate of 2 ml/min wasemployed for final purification (Lee and der Marderosian, 1988).

The single largest difficulty in detection of HPLC eluent components isthe lack of a suitable chromophore for UV detection in most saponins,although this can typically be overcome by employing techniquesincluding refractive index detection, mass detection and derivatization.

However, assuming gradient changes are small, UV detection at around203-210 nm with suitably pure solvents can generally be used. Successfulseparations also have been carried out using acetonitrile-watergradients with UV detection. Acetonitrile is preferred to methanol atlow wavelengths because of its smaller UV absorption. If the polaritydifference is not too great within a series of saponins under test (onlysmall changes in the sugar chain, for example), isocratic elution ispossible.

A useful method for separating mixtures of saponins comprises separatingon an octyl-bonded column using gradient elution with aqueousacetonitrile. The quantity of acetonitrile is increased from 30% to 40%over 20 min, yielding relatively little baseline drift under UVabsorption. More polar bidesmosidic saponins typically elute muchquicker than monodesmosidic saponins and glucuronides are less retainedthan other glycosides. An apolar octylsilyl support may be used forselection of the lipophilic part of the saponins. Using this technique,glycosides of hederagenin were eluted before the same glycosides of theless polar oleanolic acid (Domon et al., 1984).

1. Use of Derivatized Triterpenes

Detection at low wavelengths, which leads to problems of unstablebaselines caused by interference from traces of highly UV-activematerial, can be improved by HPLC analyses with derivatized triterpenes.One possibility is to functionalize free carboxyl groups found in thesaponin, as has been reported for the quantitative determination ofmonodesmosidic saponins. Treatment of oleanolic acid glycosides with4-bromophenacyl bromide in the presence of potassium bicarbonate and acrown ether results in the formation of bromophenacyl derivatives. The4-bromophenacyl derivatives strongly absorb at 254 nm and detection canbe performed at this wavelength without interference from solvent(Slacanin et al., 1988). The derivatization is as shown below.

An alternative determination method is to prepare fluorescent coumarinderivatives by esterification of the carboxylic acid moiety. By thismeans, soyasaponins were analyzed and determined quantitatively indifferent varieties and different organs of soybeans, with anthracene asinternal standard (Kitagawa et al., 1984a; Tani et al., 1985).

2. Sample Purification

In order to remove interfering material, which is often highlyUV-absorbing, a pre-purification step may be necessary. This can beachieved, for example. by use of Sep-Pak^(R) C₁₈ (Guédon et al., 1989)or Extrelut^(R) (Sollorz, 1985) cartridges.

In the case of ionic compounds, such as those containing a free carboxylgroup on the aglycone or glucuronic acid moieties, some method ofsuppressing ion formation is required if peak broadening is to beavoided. This can be achieved by addition of a low UV-absorbing acid tothe fluent, such as phosphoric acid or trifluoroacetic acid. Anotherpossibility is to use ion-pair HPLC, with a counter-ion added to themobile phase. The capacity factor of the ionic compounds is increased byforming ion complexes with the pairing reagent. Derivatization ofcarboxyl groups (as mentioned above) is an alternative to additives inthe mobile phase, resulting in considerable enhancement of peakresolution.

An advantage of quantitative HPLC over photometric methods is that theamounts of the individual saponins in a mixture or extract can bedetermined. In many instances, HPLC gives better results than thoseobtained by colorimetric, gas chromatographic and TLC-fluorimetrictechniques.

In cases where the peak resolution of saponin mixtures on reversed-phaseHPLC columns is insufficient, a number of other methods may be employedincluding utilization of hydroxyapatite columns, chemically modifiedporous glass columns, silica gel columns, and HPLC of borate complexes.

3. Hydroxyapatite

Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) is more hydrophilic than silica gel andcan be used with simple binary aqueous solvent systems, thusfacilitating detection by UV. It is stable in neutral and alkalinemedia. Recently, hard spherical particles of hydroxyapatite which areresistant to high pressure (up to 150 kg/cm²) have been prepared,broadening the applications of HPLC. Saponins differing only in theterminal pentose unit and which can not be separated by RP-HPLC can beresolved using this technique (Kasai et al., 1987b). The separation ofginsenosides from Panax ginseng (Araliaceae) was achieved in theisocratic mode (acetonitrite-water, 80:20) or, better, with a lineargradient (acetonitrile-water 70:30→90:10) (Kasai et al., 1987b). As isobserved for silica gel, the glycosides are eluted in order ofincreasing polarity, i.e., the opposite of RP-HPLC.

4. Borate Ion-Exchange HPLC

This method has found application in the analysis of mono- andoligosaccharides. The best results with this technique are obtained withan anion exchange column, for example, an Asahipak ES-502N™, 100×7.6 mincolumn from Asahi Kasei Kogyo Co. with 0.4 M H₃BO₃ in 20% (v/v)acetonitrile (pH 8) at 75° C. The chromatographic characteristics dependon the formation of borate complexes with cis-diols in the saccharidemoiety. After separations, borate can be removed as volatile methylborate by repeated co-distillation of the eluate with methanol.

5. Chemically Modified Porous Glass

Microporous glass (MPG) has a high chemical resistance and is stablebetween pH 2 and 12. Octadecyl porous glass (MPG-ODS) has been preparedas a packing for reversed-phase HPLC and used for the rapid andefficient separation of saponins. For example, it is possible toseparate both ginsenosides and saikosaponins simultaneously fromextracts of combination drugs containing ginseng and bupleurum rootusing an acetonitrile-water (25.5:74.5) mixture for the separation(Kanazawa et al., 1990a). Comparison of MPG-ODS and silica-ODS columnsfor the HPLC of ginseng extract and for mixtures of ginsenosides hasshown that the retention behavior was similar but that capacity factorswere smaller on an MPG-ODS column. The resolution of certain pairs ofginsenosides was better on MPG-ODS columns (Kanazawa et al., 1993).

6. Silica Gel

The use of water-containing mobile phases is often unavoidable for theseparation of saponins, and silica gel HPLC does not normally lenditself to such eluents. However, a modification of the column packinghas made possible the separation of water-soluble glycosides withoutcolumn deterioration. The procedure involves first washing the columnwith methanol, then with the mixture chloroform-methanol-ethanol-water(62:16:16:6) and finally the solvent system to be used for theseparation (Kaizuka and Takahashi, 1983). Using, for example, a 5 μmsilica gel columns with a water-containing eluent: hexane-ethanol-water(8:2:0.5), efficient analyses of ginseng saponins and saikosaponins fromBupleurum falcatum could be achieved.

(vii) Other Chromatographic Techniques

The isolation of pure saponins requires one or, more typically, morethan one chromatographic separation steps in order to remove other polarconstituents of alcoholic or aqueous plant extracts.

A variety of separation techniques have been described and may be usedfor separating triterpene saponins including flash chromatography, DCCC,low-pressure liquid chromatography (LPLC), medium-pressure liquidchromatography (MPLC), HPLC and conventional open-column chromatography(See, e.g., Hostettmann et al., 1986, 1991; Marston and Hostettmann,1991 b). An idea of separation conditions, solvent systems, etc. will beknown to those of skill in the art in light of the instant disclosure.The best results are usually achieved by strategies which employ acombination of methods, such as those specifically disclosed hereinbelow.

As a number of saponins are acidic, salts can form and on completion ofchromatography, treatment with an ion-exchange resin may be necessary toobtain the free saponin. Examples of suitable resin include Dowex 50W×8(H⁺ form) (Kitagawa et al., 1988; Yoshikawa et al., 1991), Amberlite IRC84 (Okabe et al., 1989; Nagao et al., 1990) and Amberlite MB-3 (Mizutaniet al., 1984). However, if neutrality or careful control of pH arenecessary to prevent decomposition, steps involving filtration onion-exchange resins should be avoided.

In certain instances, crude saponin fractions have been methylated(assuming that free COOH groups are present) in order to achievesatisfactory separations of closely related products (Okabe et al.,1989; Nagao et al., 1989, 1990).

1. Flash Chromatography

Flash chromatography is a preparative pressure liquid chromatographymethod which enables a considerable time saving when compared withconventional open-column chromatography. Ordinary glass columns are usedbut eluent is driven through a sorbent by compressed air or nitrogen,reaching a maximum pressure of about 2 bar at the top of the column. Thegranulometry of the sorbent is somewhat reduced because solvent is beingdelivered under pressure; resolution is consequently higher.

Flash chromatography can be employed as a fast alternative toopen-column chromatographic methods of preliminary fractionation. Usingthis method, separations of 10 mg to 10 g of sample can be achieved inas little as 10 min. For example, molluscicidal and fungicidalhederagenin, bayogenin and medicagenin glucosides from the roots ofDolichos kilimandscharicus (Leguminosae) were isolated with thistechnique. A methanol extract (3.3 g) was fractionated on silica gel(63-200 μm granulometry) in a 60×4 cm column with the solvent systemchloroform-methanol-water (50:10:1) at a flow rate of 15 ml/min. Thiswas sufficient to remove contaminating material and obtain twosaponin-rich fractions. The pure triterpene glycosides were obtained bya combination of DCCC and LPLC on C-8 supports (Marston et al., 1988a).

Although most applications have involved silica gel sorbents, there isan increasing trend towards RP materials. RP flash chromatographyenables the separation of saponins from other, more polar, componentssuch as oligosaccharides.

2. Low-Pressure Liquid Chromatography (LPLC)

LPLC is useful for the isolation of pure saponins because of the speedof separation and ease of manipulation. LPLC employs columns containingsorbents with a particle size of 40-60 μm. High flow rates at pressuresof up to 10 bar are possible and columns are mostly made of glass.Commercially available pre-packed columns (the ‘Lobar’ range from Merck,for example) in different sizes are ideal for the preparativechromatography of saponins in the 50-500 mg sample range. A high anduniform packing density guarantees a good separation efficiency. Stillfurther, it is relatively easy to transpose analytical HPLC conditionsonto an LPLC separation, given that the chemistry of the sorbents issimilar (Marston and Hostettmann, 1991b).

Most applications have been performed on RP sorbents, eluted withmethanol-water mixtures. It is generally only pre-purified samples whichare injected in this case. A good illustration of LPLC is provided bythe separation of molluscicidal and haemolytic oleanolic acid andgypsogenin glycosides from Swartzia madagascariensis (Leguminosae). Thedried, ground fruit pods were extracted with water and this extract waspartitioned between n-butanol and water. After open-columnchromatography of the organic phase, saponins were separated on a LobarLiChroprep C-8 column (40-63 pro; 27×2.5 cm) with methanol-water (75:25)as eluent (Borel and Hostettmann, 1987).

Joining LPLC columns in series permits an increase in loading capacityand/or separating power. This approach was used during the separation ofdammarane glycosides from Actinostemma lobata (Cucurbitaceae), whenthree Lobar 27×2.5 cm columns were connected. The eluent also containeda small amount of water (ethyl acetate-n-propanol-water 20:3:0.3)(Iwamoto et al., 1987).

3. Countercurrent Chromatography

Liquid-liquid partition methods have proved ideal for application to thefield of saponins. Very polar saponins lend themselves especially wellto countercurrent chromatographic separation, especially as there is noloss of material by irreversible adsorption to packing materials. Thisaspect has been of especial use for the direct fractionation of crudeextracts.

4. Droplet Countercurrent Chromatography (DCCC)

DCCC relies on the continuous passage of droplets of a mobile phasethrough an immiscible liquid stationary phase contained in a largenumber of vertical glass tubes. The solute undergoes a continuouspartition between the two phases. Depending on whether the mobile phaseis introduced at the top or at the bottom of these tubes, chromatographyis in the ‘descending’ or ‘ascending’ mode, respectively. The separationof closely related saponins by DCCC and even the isolation of pureproducts has been possible (Hostettmann et al., 1984). In fact, certainseparations which have not been possible by liquid-solid chromatographyhave been achieved by this technique. DCCC was capable of separatingisomeric saponins differing only in the positions of substitution ofacetate groups on the sugar residues (Ishii et al, 1984).

A number of solvent systems have been employed for the DCCC separationof saponins (see, e.g., Hostettmann et al., 1986) and, of these, thesystem chloroform-methanol-water (7:13:8) has been involved in thegreatest number of applications. Chloroform-methanol-water systems canbe used either in the ascending mode for very polar saponins or in thedescending mode for saponins possessing one or two sugars and few freehydroxyl groups.

A large-scale DCCC procedure for preliminary purification, using 18columns (30 cm×10 mm ID.) with n-butanol-saturated water as thestationary phase and water-saturated n-butanol as the mobile phase, hasbeen described (Komori et al., 1983). In some cases, two (or more) DCCCseparations are run to obtain the pure saponins.

5. Centrifugal Partition Chromatography (CPC)

The recently introduced technique of CPC holds great promise because ofits speed and versatility (Marston et al., 1990). CPC relies on acentrifugal field, produced by rotation at 800-2000 r.p.m. or faster,rather than a gravitational field for retention of the stationary phase.The principle of the method involves a continuous process ofnon-equilibrium partition of solute between two immiscible phasescontained in rotating coils or cartridges.

Instruments based on rotating coils can involve either planetary ornon-planetary motion about a central axis. One of these, the high speedcountercurrent chromatograph (HSCCC) consists of a Teflon tube of 1.6 or2.6 mm I.D. wrapped as a coil around a spool. One, two or three spoolsconstitute the heart of the instrument. In the case of cartridgeinstruments, the cartridges are located at the circumference of acentrifuge rotor, with their longitudinal axes parallel to the directionof the centrifugal force. The number and volume of the cartridges can bevaried, depending on the application to which the instrument is put.Compared with DCCC and RLCC, in which separations may take 2 days orlonger, CPC can produce the same results in a matter of hours.Instruments based on rotating coils or cartridges have capacities up tothe gram scale. A multilayer coil planet instrument has been used, forexample, for the preliminary purification of cycloartane glycosides fromAbrus fruticulosus (Leguminosae) (Fullas et al., 1990). Molluscicidaltriterpene glycosides from Hedera helix (Araliaceae) have been separatedon a different instrument, the Sanki LLN chromatograph (six cartridges;total volume 125 ml). A methanol extract of the fruit was partitionedbetween n-butanol and water. The butanol fraction was injected directlyinto the instrument in 100 mg amounts, using the lower layer of thesolvent system chloroform-methanol-water (7:13:8) as mobile phase.

The two main saponins asiaticoside and madecassoside from Centellaasiatica (Umbelliferae) have been separated with the aid of an Itomulti-layer coil separator-extractor (P.C. Inc.) equipped with a 66m×2.6 mm I.D. column (350 ml capacity), turning at 800 r.p.m. A sampleof 400 mg could be resolved with the solvent systemchloroform-methanol-2-butanol-water (7:6:3:4; mobile phase was lowerphase). Detection was by means of on-line TLC (Diallo et al., 1991). Thesame instrument was employed during the isolation of a triterpenedisaccharide from Sesamum alatum (Pedaliaceae). The lower phase of thesolvent chloroform-methanol-i-propanol-water (5:6:1:4) was chosen as themobile phase and a charge of 1.25 g was injected (Potterat et al.,1992),

6. Combination of Methods

It is rare that a single chromatographic step is sufficient to isolate apure saponin from an extract. As a general rule, several preparativetechniques are required in series to obtain the necessary product. Acombination of classical techniques (such as open-column chromatography)and modern high-resolution methods (such as HPLC) has proved suitablefor the separation of many saponins.

For example, a combination of MPLC on silica gel and RP material, LPLCand centrifugal TLC for separation of saponins (Hamburger andHostettmann, 1986). Similarly, the isolation of five triterpene saponinsfrom Swartzia madagascariensis (Leguminosae) required open-columnchromatography, LPLC and MPLC (Borel and Hostettmann, 1987).

CPC has been used in conjunction with flash chromatography and OPLC forthe isolation of triterpene glycosides from Abrus fruticulosus(Leguminosae). A multilayer coil instrument (solventchloroform-methanol-water 7:13:8, lower phase as mobile phase) providedinitial purification, while flash chromatography and OPLC were effectivefor obtaining the pure substances (Fullas et al., 1990).

The straightforward combination of flash chromatography on unmodifiedsilica gel with either flash chromatography or open-columnchromatography on RP material can sometimes be sufficient for thepurification of saponins (Schöpke et al., 1991).

Another strategy involves passing extracts (after preliminary partition)over highly porous polymers and following this step by furtherfractionation of the crude saponin mixtures. This approach was used inthe isolation of 3β-hydroxyolean-12-en-28,29-dioic acid glycosides fromNothopanax delavayi (Araliaceae). A methanol extract of the leaves andstems was partitioned between hexane and water. The aqueous layer waschromatographed on a Diaion HP-20 column and eluted with water, 10%methanol, 50% methanol, 80% methanol, methanol and chloroform. Theglycosides were obtained by subsequent column chromatography of the 80%methanol eluate on silica gel with ethyl acetate-ethanol-water (7:2:1)(Kasai et al., 1987a). For the isolation of triterpene andnon-triterpene saponins from Acanthopanax senticosus (Araliaceae), theprocedure began with a fractionation of the methanol extract of theleaves on Diaion HP-20 polymer. The fraction eluted with methanol waschromatographed on silica gel (chloroform-methanol-water 30:10:1) andall the resulting fractions were subjected to column chromatography onLiChroprep RP-8. Final purification was achieved by HPLC on TSK-GELODS-120T (300×21 min; methanol-water 70:30; 6 ml/min; RI detection) orchromatography on a hydroxyapatite column (acetonitrile-water 85:15)(Shao et al., 1988).

A procedure for separation of oleanic acid glycosides comprisesemploying a combination of Sephadex LH-20 (methanol), DCCC(chloroform-methanol-water 7:13:8) and HPLC (C-18, methanol-water 65:35)(De Tommasi et al., 1991).

(viii) Color Reactions

Reactions of triterpenes with any of a variety of agents may be used toproduce colored compounds for the quantitative or qualitativedetermination of triterpenes. For example, aromatic aldehydes such asaisaldehyde and vanillin in strong mineral acid, for example, sulfuric,phosphoric, and perchloric acids, give colored products with aglycones,having absorption maxima between 510 and 620 nm. In these reactions, adehydration is believed to occur, forming unsaturated methylene groupswhich give colored condensation products with the aldehydes. Withvanillin-sulfuric acid, triterpene saponins with a C-23 hydroxyl grouphave a peak located between 460 and 485 nm (Hiai et al., 1976).

Unsaturated and hydroxylated triterpenes and steroids give a red, blueor green coloration with acetic anhydride and sulfuric acid (Abisch andReichstein, 1960). Since terpenoid saponins tend to produce a pink orpurple shade and steroid saponins a blue-green coloration,differentiation of the two classes is possible using this technique.

A large number of other agents may be used for detection of triterpenesincluding: cerium(IV) sulphate or iron (III) salts and inorganic acids,such as sulfuric acid, which give a violet-red coloration of thesolution; a 30% solution of antimony(III) chloride in aceticanhydride-acetic acid reagent, which gives color reactions withhydroxytriterpenes and hydroxysteroids; antimony(III) chloride innitrobenzene-methanol, which can be used to differentiate the5,6-dehydro-derivatives of steroid glycosides (diosgenin and solasodineglycosides) and 5α- or 5β-H-derivatives (e.g., tomatine); and carbazole,which in the presence of borate and concentrated sulfuric acid willindicate the presence of uronic acids (Bitter and Muir, 1962).

Exemplary reagents for detection and for the spectrophotometric andcolorimetric determination of saponins are listed below, in Table 2.

TABLE 2 Visualization Reagents for Triterpene Saponins Reagent ReferenceVanillin-sulfuric acid Godin, 1954 Vanillin-phosphoric acid Oakenfull,1981 Liebermann-Burchard (acetic Abisch and Reichstein, 1960anhydride-sulfuric acid) 1% Cerium sulphate in 10% sulfuric acid Wagneret al., 1984 10% Sulfuric acid in ethanol Kitagawa et al., 1984b 50%Sulfuric acid Price et al., 1987 p-Anisaldehyde-sulfuric acid Price etal., 1987 Komarowsky Wagner et al., 1984 (p-hydroxybenzaldehyde-sulfuricacid) Antimony (III) chloride Wagner et al., 1985 Blood Wagner et al.,1984 Water Wagner et al., 1984

(ix) Isolation of Triterpene Glycosides from Acacia Victoriae

Legume extracts were prepared by chloroform:methanol or dichloromethane:chloroform extraction at The University of Arizona (Tucson, Ariz.). Theinventors isolated mixtures of triterpene glycosides from Acaciavictoriae (Benth.) (Leguminosae). The first collection ofUA-BRF-004-DELEP-F001 was processed as follows: (1) grinding to 3 mmparticle size in Wiley mill, (2) packing into two-liter percolationunit, (3) extracting the ground biomass with dichloromethane:methanol(1:1) for 4 hr. followed by overnight and the combined fractions weredried in vacuo to generate UA-BRF-004-DELEP-F001 (52 g). F001 (51.5 g)was extracted with ethyl acetate to yield active insoluble (34.7 g)material designated as F004. Flash chromatography using 1.7 kg of silicagel (Merck, 23-220 micron particle size) was used to fractionate F004(34.2), 51 670-ml fractions eluted with dichloromethane: methanol(step-gradient-95-0%: methanol 5-100%). the Column was washed withnine-liters of methanol followed by six-liters of methanol:water (80:20)and then six-liters of same eluent with 1% formic acid added. Based onTLC fractions 23-34 and 39-40 were combined to 17.2 g of F023. MediumPressure Liquid Chromatography (MPLC, Buchi 632 system) was used twicewith 8 g of F023 each on a 4.9×46-cm column packed with Lichroprep C18,15-25 micron particle size using step gradient of acetonitrile: water(0,10,20,30,50% acetonitrile in water) followed by 100% methanol wash.Of the 16 g 0-20% acetonitrile, yield was seven grams of F027, which wasinactive. The remaining material was combined and subjected torepetitive MPLC with the same system using 30-40% acetonitrile tominimize overlap and generate fractions F028-F036. Although most ofthese fractions demonstrated antitumor activity, F035 (Fraction 35)(highest yield of 2.19 g) was selected for further testing andevaluation.

III. Structural Determination of Triterpenes

Various methods may be employed for the qualitative and quantitativedetermination of triterpenes and their activities including: piscicidalactivity, gravimetry, spectrophotometry, TLC, GC, HPLC, HMQC, HMBC,NOESY, COSY, NMR, X-Ray crystallography etc. Determinations based onclassical properties of triterpene saponins (surface activity, fishtoxicity) have largely been replaced by photometric methods such asdensitometry, colorimetry of derivatives and, more recently, by GC, HPLCand particularly, NMR. Spectrophotometric methods are very sensitive butnot typically suitable for estimating triterpenes in crude plantextracts since the reactions are not specific and colored products mayform with compounds which accompany the triterpenes, such asphytosterols and flavonoids. Another problem, common to much of theanalytical work on saponins, is their incomplete extraction from theplant material. However, a number of techniques are widely availablewhich are suitable for quantitating triterpenes.

There are several basic problems to be solved in the structureelucidation of saponins: the structure of the genuine aglycone; thecomposition and sequence of the component monosaccharides in thecarbohydrate moiety; how the monosaccharide units are linked to oneanother; the anomeric configuration of each glycosidically linkedmonosaccharide unit; and the location of the carbohydrate moiety on theaglycone.

The necessary approach is to apply a combination of methods in order toarrive at a final conclusion for the structure. Structural studies areusually a stepwise process, in which the saponin is gradually brokendown into smaller fragment's which themselves are analyzedspectroscopically. By a judicious handling of the data from thefragments, an idea of the composition of the saponin is derived.

The quantities of pure saponins isolated are often small, thus the useof highly sensitive, high-resolution and, if possible, non-degradativemethods is preferable in order to aid the structure determination of asaponin. Innovations in NMR spectroscopy and mass spectrometry (MS) haveprovided such necessary abilities for the investigations of complexsaponins. Through combinations of these and other techniques, structuraldeterminations can be made. For example, FAB-MS gives information aboutthe molecular weight and, in many cases, the sugar sequence, while I-Dand 2-D NMR techniques permit the localization of sugar linkages andcontribute to the structure elucidation of the aglycone. Such structuraldetermination and chemical studies have been thoroughly discussed in areview by Tanaka and Kasai (1984).

(i) Nuclear Magnetic Resonance (NMR)

Of all the modern methods for the structure elucidation ofoligosaccharides and glycosides, NMR spectroscopy provides the mostcomplete information, with or without prior structural knowledge(Agrawal, 1992). It is the only approach which can, in principle, give acomplete structure without resort to any other method.

1. ¹³C-Nuclear Magnetic Resonance

Carbon-13 NMR spectroscopy, now widely used for the structuredetermination of saponins, is a fast and non-destructive method butrequires quite large quantities of sample (mg amounts). Analysis of thespectra allows conclusions to be drawn about positions of attachment ofthe glycosidic chains to the aglycone; the sequence, nature and numberof monosaccharides; configuration and conformation of theinterglycosidic linkages; the presence of acylglycosides in the chains;the nature of the aglycone; and the structures of attached ester acids.

For assigning chemical shifts, it is helpful to compare observed datawith data reported for model and related compounds. As a guide to someof the typical chemical shifts in the ¹³C-NMR spectrum of a triterpenesaponin, one may use the known shifts of the bayogenin glycoside (Domonand Hostettmann, 1984). Additionally, compilations of assignments of¹³C-NMR signals for oleanane (Patra et al., 1981; Agrawal and Jain,1992), ursane, lupane (Wenkert et al., 1978; Sholichin et al., 1980),hopane (Wenkert et al., 1978; Wilkins et al., 1987) and lanostane(Parrilli et al., 1979) triterpenes have been made (Nakanishi et al.,1983). The relevant data for dammarane glycosides have been summarizedin a review (Tanaka and Kasai, 1984), while ¹³C-NMR spectroscopy ofsaikogenins (Tori et al., 1976a) and of saikosaponins (Tori et al.,1976b) has been described. Ginseng sapogenins and related dammaranetriterpenes also have been studied (Asakawa et al., 1977). ¹³C-NMRspectroscopy of acacic acid has also been described (Kinjo et al.,1992).

In ¹³C-NMR, when hydroxyl groups are derivatized, i.e. glycosylated,methylated (or acetylated), the α- and β-carbons of both the sugar andaglycone moieties undergo characteristic shifts. For example, the α-CHsignals are shifted downfield, while the β-C signals are shiftedupfield, a shift resulting from the general γ-upfield shift). Thus,glycosylation of an aglycone causes a downfield shift of the α-carbonand an upfield shift (glycosidation shifts) of the adjacent carbon atoms(Tori et al., 1976b; Kasai et al., 1977). In oleananes, glycosidation ofthe 3β-OH group causes C-3 to shift downfield by c. 8.0-11.5 p.p.m., C-2and C-4 to shift by +0.9 or −0.9 to −1.9 p.p.m., C-23 to shift upfieldby 0.5-5.1 p.p.m. and C-24 to shift by −0.2 to 1.6 p.p.m. Glycosidationof the 28-COOH group causes the carboxylic carbon resonance to moveupfield (2.5-5.0 p.p.m.) and the C-17 signal to move downfield (1.0-2.5p.p.m.) (Agrawal and Jain, 1992). A comparison of the ¹³C-NMR data ofthe aglycone and saponin, therefore, gives the site of the sugar linkage(Seo et al., 1978; Tanaka, 1985).

In a similar fashion, ¹³C-NMR will give an indication (in simplersaponins) of interglycosidic linkages by considering displacements ofchemical shifts when compared with model compounds (Konishi et al.,1978). Carbon-13 NMR data for methyl β-D-fucopyranoside have beentabulated by Seo et al., (1978), while ¹³C-NMR signals for the morecomplex sugars are listed by Gorin and Mazurek (1975) and Dorman andRoberts (1970). Apiose gives characteristic ¹³C-NMR signals and thesehave been documented (Sakuma and Shoji, 1982; Adinolfi et al., 1987;Reznicek et al., 1990).

2. ‘H-Nuclear Magnetic Resonance

Although ¹³C-NMR spectral analysis and signal assignment has become aparticularly useful procedure in the structure determination ofsaponins, the complete assignment of their ¹H-NMR spectra has onlyseldom been reported. The ¹H-NMR spectra have characteristically provedcomplex and tedious to analyze. The vast majority of proton resonancesof the carbohydrate moiety appear in a very small spectral width of3.0-4.2 p.p.m., with subsequent problems of overlapping. These derivefrom the bulk of non-anomeric sugar methine and methylene protons whichhave very similar chemical shifts in different monosaccharide residues.

However, the methyl peaks of triterpenes are readily discernible andmost proton resonance positions in oleanene, ursene and relatedskeletons have been assigned since the 1960s (Kojima and Ogura, 1989) bya variety of techniques. For example, the complete ¹H- and ¹³C-NMRspectral assignments of soyasapogenol B (33) and the configuration ofthe C-4 hydroxymethyl substituent have been established by a combinationof ¹³C-DEPT, ¹³C-APT, 2-D correlation spectroscopy (COSY) (¹H-¹³C-COSY,¹H-¹H COSY) and ¹H-¹H ROESY (2-D nuclear Overhauser enhancement (NOE) ina rotating frame) techniques (Baxter et al., 1990). The assignments ofquaternary carbon resonances in this sapogenin have been confirmed by¹H-detected heteronuclear multiple-bond (HMBC) and one-bond (HMQC)spectroscopy (Massiot et al., 1991b). A full interpretation of the¹H-NMR spectra of diosgenin and solasodine has also been achieved (Puriet al., 1993).

Some useful data can be obtained from ¹H-NMR spectra for the anomericconfigurations and linkages of the sugar chain. For example, thecoupling constant of the C-1 proton of α-linked glucose units isapproximately 3 Hz, while β-linked units have a coupling constant of 6-7Hz. More details on the coupling constants of anomeric sugar protons canbe found elsewhere (Lemieux et al., 1958; Capon and Thacker, 1964; Kizuand Tomimori, 1982).

When difficulties arise in determining configurations of hydroxyl groupsat C-2, C-3 and C-23, C-24 of oleanene and ursene triterpenes, analysisof the ¹H-NMR signal peaks of the protons on oxygen-bearing carbon atomsgives valuable information (Kojima and Ogura, 1989).

(ii) 1-D and 2-D NMR Techniques

In practice, certain ¹H and ¹³C NMR spectra can be identified andassigned on the basis of shift arguments, but for interpreting theresults of NMR studies in a rigorous manner, an NMR spectrum should beassigned unambiguously, which means establishing which peaks areassociated with which carbon and/or hydrogen in the structure. Thisinformation, in most cases, cannot be obtained from one-dimensional ¹Hand ¹³C NMR spectral data, but can better be determined with the aid oftwo-dimensional studies. These studies simplify spectral analysis byspreading out information into two frequency domains and by revealinginteractions between nuclei. Despite the fact that the mechanisms onwhich the various pulse sequences are established may be intricate, theinterpretation of two-dimensional NMR spectra is usuallystraightforward. A large number of different two-dimensional NMR studieshave been devised to solve chemical structures. Examples of suchtechniques, as well as other NMR techniques specifically contemplated bythe inventors for use in the chemical elucidation of the triterpenesaponins of the invention, are described below, and in Table 3.

1. HMBC, HMQC

The use of HMQC and HMBC ¹³C multiple-quantum coherence spectra isvaluable not only for aglycone assignments, but also for sugar sequencedetails. The use of HMBC and HMQC is analogous to ¹³C-¹H heteronuclearcorrelated spectroscopy (HETCOR), but instead of observing ¹³C, the moreabundant ¹H is detected. For example, in the case of bellissaponins fromBellis perennis (Asteraceae), it was possible to assign all the chemicalshifts in the ¹H-NMR spectrum by considering ¹³C-NMR data in conjunctionwith 2-D ¹H-detected HMQC and HMBC spectra. Cross peaks corresponding totwo and three bond couplings were observed for nearly all possiblecorrelations in the molecule. Similarly, long-range ¹H-¹³C correlationsin HMQC and HMBC spectra may be used for the determination of thesequence and positions of attachment of sugar moieties (Schopke et al.,1991).

2. 2-D-NOESY

This technique has been applied, for example, in the determination ofthe sugar sequence of cyclamiretin A glycosides (ardisiacrispins A andB) (Jansakul et al., 1987) and the monosaccharide sequence ofsaxifragifolin A from Androsace saxifragifolia (Primulaceae) (Waltho etal., 1986). The location of rhamnosyl and glucosyl linkages on thearabinose moiety of kalopanax saponin C were confirmed by NOESY aftersugar sequence analysis of the permethylated saponin. Cross peaks wereobserved between H-1 of a rhamnosyl moiety and H-2 of an arabinosylmoiety, as well as between H-1 of the glucosyl moiety and H-3 of thearabinosyl moiety (Shao et al., 1989b). The structures of the sugarmoieties of furostanol saponins from Balanites aegyptiaca (Balanitaceae)have been elucidated by means of 2-D NOESY on a 400 MHz NMR instrument(Kamel et al., 1991).

The concerted use of 2-D NMR techniques led to complete ¹³C and ¹Hassignments for the oligosaccharide segment of the sarsasapogeninglycoside3-O-[{α-L-rhamnopyranosyl(1→4)}{β-D-glucopyranosyl(1→2)}-⊖-D-glucopyranosyl]-(25S)-5β-spirostan-3β-ol.A combination of DEPT, HETCOR, long-range HETCOR, different homonucleartechniques, NOESY and INEPT were applied to the structure elucidation inorder to resolve problems caused by overcrowding of the proton spectrum(Pant et al., 1988d).

The identification and sequencing of sugars in the pentasaccharidesaponin3-O-[β-D-xylopyranosyl(1→3)-α-L-arabinopyranosyl](1→4)-β-D-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-L-arabinopy-ranosyl]-hederageninfrom Blighia welwitschii (Sapindaceae) was possible by NMR techniquesalone, using a 500 MHz instrument. The saponin was first acetylated, andsubsequent analysis of the DQF-COSY, NOESY and ROESY spectra allowedassignment of structure. Information obtained from NOE data was mosthelpful for establishing the sugar sequence (Penders et al., 1989).

A saponin containing ten sugar residues from Solidago gigantea(Asteraceae) was identified by NMR, based on multi-step RCT studies.This involved COSY, heteronuclear COSY, COSY-type H-H-C coherencetransfer and 2-D NOESY studies. Extensive degradation studies were thusavoided and structure determination was possible with 30 mg of theproduct (Reznicek et al., 1989a; 1989b). Similar techniques wereemployed for the structure determination of another four glycosides,giganteasaponins 1-4 (bidesmosides of bayogenin containing nine or tensugar units), from the same plant (Reznicek et al., 1990a).

A combination of 2-D COSY, HMBC and ROESY NMR studies was sufficient togive the sequence and linkage positions of the hexasaccharide inmimonoside A, an oleanolic acid saponin from Mimosa tenuiflora(Leguminosae) (Jiang et al., 1991).

The 2-D NMR of peracetylated and underivatized chrysantellin A hasallowed the assignment of protons and the sequencing of sugars. Theesterified xylose moiety was shown to exist in the β-form and have a ¹C₄conformation. Among the techniques employed were HMQC, HMBC and ROESY(or, more precisely, CAMELSPIN (cross-relaxation appropriate forminimolecules emulated by locked spins)) on the peracetylated derivativeand HOHAHA, TOCSY on the native saponin. The ROSEY study wasparticularly useful for determining the sugar sequence (Massiot et al.,1991a).

The sequences of sugar and interglycosidic linkages of triterpeneglycosides from marine organisms have been established from NT₁ data andNOESY studies (Miyamoto et al., 1990) but this methodology is limited bythe complexity of the ¹H-NMR spectra in the 3-5 p.p.m. region, whichusually precludes the measurement of NOE for a large number of protons.However, a combination of COSY, NOESY and direct and XHCORR NMRspectroscopy has allowed complete signal assignment and structuralanalysis of pentasaccharide triterpene saponins from the sea cucumberHolothuria forskalii (Rodriguez et al., 1991).

In the structure determination of santiagoside, an asterosaponin fromthe Antarctic starfish Neosmilaster georgianus, the techniques of COSY,TOCSY, HMQC and ROESY NMR spectroscopy were extensively applied, withROESY studies being used to resolve the exact sequence of sugars, theirpoints of attachment and the stereochemistry (Vazquez et al., 1992).

3. COSY

There are two fundamental types of 2-D NMR spectroscopy: J-resolvedspectroscopy in which one frequency axis contains spin coupling (J) andthe other chemical shift information, and correlated spectroscopy inwhich both frequency axes contain chemical shift (δ) information(Agrawal, 1992). One of the major benefits of 2-D analysis is that itprovides a method of overcoming the problem of spectral crowding. Inhigh-field ¹H-COSY this is especially true of the 2.5-4.0 p.p.m. region,thus simplifying the assignment of saccharide protons. Under favorableconditions, all the protons present in a given sugar residue can beidentified.

Several general conclusions may be drawn from COSY spectra. For example,substitution positions of monosaccharide units can be determined by thepresence or absence of a corresponding hydroxyl proton; ring sizes ofthe monosaccharides can be determined directly; and the nature of thecross-peaks reveals the multiplicity of overlapping peaks providing anestimate of coupling constants.

In certain cases, structure elucidation of a saponin, together with itssugar sequence, has been achieved by ¹H-NMR 1-D and 2-D spectroscopyalone (Massiot et al, 1986; 1988b). The saponin is first peracetylatedand if the field strength is sufficient (>300 MHz), the sugar protonresonances split into two zones: one between 4.75 and 5.40 p.p.m.assigned to CHOAc and the other between 3.0 and 4.3 p.p.m. assigned toCH₂OAc, CHOR and CH₂OR. Anomeric protons are located between these twozones in the case of ether linkages or at a higher frequency than 5.5p.p.m. for ester linkages.

Peracetylation also gives derivatives which are soluble in chloroform,benzene or acetone. In the equivalent perdeuterated solvents, themobility of the molecules is such that signals are observed more clearlyand coupling constants can be measured with high accuracy. For theacetylated alfalfa root saponin, COSY and long-range COSY studies weresufficient to identify the structure as Ara-²Glc-²Ara-³hederagenin²⁸-Glc(Massiot et al., 1986).

The structures of further peracetylated saponins from the leaves ofalfalfa, Medicago sativa (Leguminosae) and from Tridesmostemonclaessenssi (Sapotaceae) have been elucidated by similar techniques tothose outlined above. Confirmation of assignments and sugar sequenceswas obtained from HMQC (for ¹J couplings) and HMBC (for ²J and ³Jcouplings) and homonuclear Hartmann-Hahn (HOHAHA) triple relayed COSYand ROESY studies (Massiot et al., 1990; 1991b). The ester sugar chainsof the saponins from T. claessenssi contain a β-D-xylose moiety in theunusual ¹C₄ configuration (all the substituents are axial). At 600 MHz,the ¹H-NMR spectrum may be sufficiently well resolved to allowassignment of all ¹H chemical shifts without peracetylation (Schöpke etal., 1991).

4. Long-Range COSY

This technique has been employed for the assignment of sugar protons inthe steroid saponins from Allium vineale (Liliaceae) (Chen and Snyder,1987; 1989). Long-range ¹H-¹³C COSY has also been used for aglyconestructure determination in a cycloastragenol saponin (Wang et al.,1989b) and for the location of a ⁴J inter-sugar coupling between theanomeric proton of the inner glucose and H-2 of the inner arabinose ofthe Medicago sativa saponin described above (Massiot et al., 1986).

5. Double Guantum Filtered, Phase-Sensitive COSY (DQF-COSY, DQ-COSY)

This technique was applied to the assignment of sugar protons in Alliumsteroid saponins (Chen and Snyder, 1987; 1989) and to the assignments of¹H chemical shifts in the 16α-hydroxyproto-bassic acid glycosides fromCrossopteryx febrifuga (Rubiaceae) roots (Gariboldi et al., 1990). Thesame technique was used to provide a complete assignment of saccharideprotons in the acetylated hederagenin derivative from Sapindus rarak(Sapindaceae) fruits (Hamburger et al., 1992). Interglycosidic linkageswere established by NOE difference spectroscopy (Hamburger et al.,1992).

6. HOHAHA

The proton coupling networks of aglycones of gypsogenin and quillaicacid glycosides have been completely elucidated by HOHAHA studies. Theseare similar to COSY studies (and related to total correlationspectroscopy—TOCSY) except that the observed correlation cross peaks arein phase, thereby preventing accidental nulling of overlapping peaks.For the elucidation of carbohydrate chains, vicinal coupling constantsextracted from HOHAHA studies allows the determination of the relativestereochemistry of each asymmetric center, thus enabling identificationof the monosaccharides. Heteronuclear H-C relay studies may be used forassignment of ¹³C resonances in the saccharide moieties and the sugarlinkages determined from HMBC spectra (Frechet et al., 1991).

7. FLOCK, COLOC and NOE

Long-range heteronuclear correlation spectroscopy incorporating bilinearrotation decoupling pulses (FLOCK) has been used for the observation of¹H-¹³C long distance couplings in alatoside A from Sesamum alatum(Pedaliaceae). Thus, interactions between the proton at C-18 and thecarbon atoms C-13, C-17 and C-28 were observed. In conjunction withlong-range hetero-nuclear ¹³C-¹H correlation (XHCORR), much informationwas gathered about the structure of the novel seco-ursene aglycone(Potterat et al., 1992).

An example of ¹³C-¹H 2-D correlation spectroscopy (COLOC) optimized forlong-range couplings (²J_(CH) and ³J_(CH)) is to be found in thestructure elucidation of saponins from Crossopteryx febrifuga(Rubiaceae) (Gariboldi et al., 1990).

NOE has found extensive use in the structure determination of saponins,for example, in the assignment of saccharide protons and sugar sequenceof luperoside I (Okabe et al., 1989) and camellidins I and II (Nishinoet al., 1986). A NOE between the H-2 of arabinose and the anomericproton of rhamnose helped to confirm the Rha-²Ara-disaccharide linkagein ziziphin (Yoshikawa et al., 1991b). The method has wide applicationssince connectivities are often observed between the anomeric proton andthe aglycone proton at the linkage position. Negative NOE have beenobserved between the proton at the C-3 position and the anomeric protonof the 3-O-glycoside residue in cycloastragenol and other saponins (Wanget al., 1989b).

TABLE 3 Selected NMR Approaches for Use in the Structure Establishmentof Triterpene Saponins NMR Study (Acronyms) Comments Attached protontest (APT), Distortionless Discriminates among carbon types; enhancementby polarization transfer (DEPT), Spectral editing Insensitive nucleienhancement by polarization transfer (INEPT) Incredible naturalabundance double-quantum ¹³C-¹³C connectivity, establishment transferstudy (INADEQUATE) of molecular skeleton ¹H, ¹H-COSY Homonuclear shiftcorrelation a) normal Elucidation of direct couplings b) with delaysDetection of small couplings c) double-quantum filtered-(DQF) - COSYDetermination of vicinal and geminal coupling constants d) ExclusiveCOSY (E. COSY) Accurate determination of J e) Geminal COSY (Gem - COSY)Identification of geminal spin systems f) Triple-quantum filtered(TQF) - COSY Detection of three or more mutually coupled spin systemsRelayed coherence transfer (RCT), Total correlation Identification ofall protons (TOCSY), and Hartmann-Hahn study (HOHAHA) belonging to asingle spin system; Coherence transfer across scalar connectivity(particularly useful in identifying monosaccharide residues) Homonuclearnuclear Overhauser and exchange Identification of protons that arespectroscopy (NOESY and ROESY) within 5A of one another (¹H, ¹Hcorrelation through space); Stereochemical analysis (orientation ofsubstituents); Intra- and inter-residual connectivities (sequenceanalysis in sugar chain including sugar - aglycone linkage)1H{^(13c)C}SBC (HETCOR and HMQC) Heteronuclear shift correlation;Assignments of directly bonded ¹H and ¹³C shifts HMQC-TOCSY andHMQC-RELAY Cross assignments of ¹H and ¹³C shifts 1H{¹³C}MBC (Long-rangeHETCOR and HMBC) Assignment of quaternary C; Correlation of a protonresonance with a carbon resonance 2-4 bonds distant; Intra- andinter-residual assignments (inter-glycosidic and sugar-aglyconelinkage); Confirmation of molecular structure

(iii) Spectroscopic and Other Techniques for Structure Elucidation

The structure elucidation of saponins and the corresponding aglyconesrelies not only on chemical methods but also on spectroscopic andrelated techniques, e.g., IR, UV, NMR, MS, optical rotary dispersion(ORD), circular dichroism (CD), and X-ray analysis. Modern advances insome of these techniques, most notably in NMR spectroscopy and MS, havefacilitated the task of analyzing saponins and their correspondingfragments from cleavage reactions, such that the information can becollated and the relevant structures determined. Furthermore, NMRspectroscopy is a non-destructive technique and both NMR and MS allowexamination of the intact saponin.

An integrated approach for solving saponin structures is necessary, withthe different spectroscopic techniques each providing a certaincontribution to the ensemble of data.

1. Mass Spectrometry (MS)

The choice of ionization method in MS depends on the polarity, liabilityand molecular weight of the compound to be analyzed. It is principallythe so-called ‘soft’ ionization techniques such as FAB anddesorption/chemical ionization (D/CI) which are employed to obtainmolecular weight and sugar sequence information for naturally occurringglycosides (Wolfender et al., 1992). These permit the analysis ofglycosides without derivatization. In certain cases, fragmentations ofaglycones are observed, but electron impact mass spectra (EI-MS) aremore useful for this purpose.

2. Fast Atom Bombardment MS (FAB-MS)

In FAB studies, an accelerated beam of atoms (or ions) is fired from agun towards a target which has been preloaded with a viscous liquid (the‘matrix’-usually glycerol or 1-thioglycerol) containing the sample to beanalyzed (Barber et al., 1981; 1982). When the atom beam collides withthe matrix, kinetic energy is transferred to the surface molecules, alarge number of which are sputtered out of the liquid into the highvacuum of the ion source. Ionization of many of these molecules occursduring the sputtering, giving both positive and negative ions. Eithercan be recorded by an appropriate choice of instrumental parameters butnegative ions have proved more useful, on the whole, for saponin work.

3. Secondary Ion Mass Spectrometry (SIMS)

This is another particle-induced desorption technique, in which keV ionsimpinging on the surface of a thin film of biomolecule induce the samedesorption ionization as in PD-MS (Benninghoven and Sichtermann, 1978).The utility of this method in the structural investigation of three newbidesmosides, acetyl-soyasaponins A₁, A₂ and A₃, isolated from Americansoybean seeds (Glycine max, Leguminosae) has been demonstrated. Thesignificant fragment ion peaks provided information regarding the modeof acetylation in the monosaccharide units, as well as the sequence ofthese units (Kitagawa et al., 1988).

4. Laser Desorption (LD)

In LD it has been demonstrated that excitation by short duration laserpulses (<10 ns) produces patterns of desorbed molecular ions similar toPD and SIMS. Laser desorption/Fourier transform mass spectrometry(LD/FTMS), a technique which also is suitable for the analysis ofcomplex glycosides, produces spectra which are different from andcomplementary to FAB-MS.

5. Field Desorption MS (FD-MS)

This technique is practical for determining the molecular weights ofsaponins, together with the number, nature and sequence of sugarresidues (Komori et al., 1985). However, the experimental complexity ofFD-MS and the fact that FAB-MS produces longer-lasting spectra has meantthat the FD-MS approach has decreased in popularity recently. FD massspectra have the added drawback that they are complicated by thepresence of cationized fragments, making interpretations difficult. Allthe same, FD-MS has been very successfully applied to the structureelucidation of saponins (Hostettmann, 1980).

(iv) Liquid Chromatography-Mass Spectrometry (LC-MS)

Several types of efficient interfaces for direct and indirectintroduction of HPLC column effluent for mass spectrometry analysis havenow been developed. For example, qualitative analysis of crude saponinfractions has been carried out by combining semi-micro HPLC with aflit-fast atom bombardment (FRIT-FAB) interface (Hattori et al., 1988).For this application, an NH₂ column, (e.g., μS-Finepak SIL NH₂, Jasco;25 cm×1.5 mm internal diameter (I.D.)) is used, rather than an octadecylsilica column, with a 1:20 split ratio of effluent (100 μ/min→5 μl/min).Elution with a linear gradient of acetonitrile and water containing 1%glycerol will typically allow a better peak sharpness than that obtainedby isocratic elution. Negative FAB mass spectra have been recorded forsaponins with a molecular weight of up to 1235. Pseudomolecular [M−1]−ions as well as fragment ions ascribed to the cleavage of sugar moietieswere observed with this technique (Hattori et al., 1988).

A FRIT-FAB LC-MS system has also been described for the separation of amixture of the isomeric saponins rosamultin and arjunetin (bothmolecular weight 650) from Rosa rugosa (Rosaceae). Rosamultin (an ursaneglycoside) and arjunetin (an oleanane glycoside) both have a singleglucose residue at C-28 and were analyzed in both the negative andpositive FAB modes with xenon as neutral gas. HPLC was performed on anoctadecylsilica column (250×1.5 mm) with acetonitrile-water (7:3,containing 0.5% glycerol) as solvent at a flow rate of 1 ml/min.Pseudomolecular [M+1]− and [M+1]+ ions were observed, together withstrong peaks caused by the parent aglycones in the negative FAB massspectra (Young et al, 1988).

It also is possible to detect saponins by dynamic secondary ion massspectroscopy (SIMS), a technique similar to dynamic FAB interfacing inwhich eluent is passed directly into the source. Thus, HPLC combinedwith UV (206 nm) and SIMS detection has been employed to analyze amixture of one mono- and two bidesmosidic triterpene glycosides (Marstonet al., 1991).

A disadvantage with interfaces of the FRIT-FAB and CF-FAB type is thelow flow rate required (around 1-5 μl/min). After HPLC separation,effluent splitting is necessary. The thermospray (TSP) interface(Blackley and Vestal, 1983), however, is characterized by its simplicityand its ability to handle flow rates of 1-2 ml/min. This makes thetechnique more attractive for problems involving the analysis of plantconstituents. At the heart of the TSP technique is a soft ionization ofmolecules, similar to chemical ionization MS. This allows analysis ofnon-volatile and thermally labile mono-, di- and even triglycosides.Information is provided about the molecular weight of the saponin andthe nature and sequence of the sugar chains. TSP LC-MS has been used forthe analysis of molluscicidal saponins in a methanol extract ofTetrapleura tetraptera (Leguminosae) fruits (Maillard and Hostettmann,1993). With post-column addition of 0.5 M ammonium acetate (0.2 ml/min)to provide the volatile buffer for ion evaporation ionization, the TSPLC-MS total ion current (mass range 450 to 1000 a.m.u) corresponded wellwith HPLC-UV analysis at 206 nm. Ion traces at m/z 660, 676, 880 and 822gave signals representing the pseudomolecular [M+H]+ ions of the majorsaponins. The TSP mass spectrum acquired for each saponin in the extractdisplayed a major peak for the pseudomolecular [M+H]+ ion.Fragmentations of the sugar moieties were observed for the principalmolluscicidal saponin aridanin, where loss of a N-acetylglucosyl moietygave rise to an [A+H]+ peak for the aglycone (Maillard and Hostettmann,1993).

LC-MS, as applied to the investigation of saponins, has great potentialutility as GC-MS is of minimal practical use and in HPLC alone theidentities of peaks can only be confirmed by their retention times. Notonly is LC-MS amenable to the analysis of triterpene glycosides in plantextracts but it will also be of value, via MS-MS, for the structuredetermination of individual saponins in the extracts.

(v) Infrared Spectroscopy (IR)

Apart from the usual applications of IR, there are one or two featureswhich are of particular relevance to the structure elucidation ofsaponins. IR is useful for the characterization of steroid sapogeninsbecause several strong bands between 1350 and 875 cm⁻¹ are diagnosticfor the spiroketal side chain (Jones et al., 1953). Four bands, 980 (Aband), 920 (B band), 900 (C band) and 860 cm⁻¹ (D band) have beenassigned as characteristic of the E and F rings. With 25R-sapogenins theB band has a stronger absorbance than the C band, while in the25R-series this relationship is reversed. In sapogenins having oxygensubstituents in the E and F rings or at position 27, the four bands areconsiderably changed (Takeda, 1972).

The presence of ionized carboxyl groups in saponins can be ascertainedby bands in the IR spectrum at 1610 and 1390 cm⁻¹ (Numata et al., 1987).This information is useful during the isolation procedure, when it isimportant to know whether carboxyl groups in the molecule are ionized.

(vi) X-Ray Crystallography

X-ray crystallography has been used to elucidate the molecular geometryof the trisaccharide triterpene asiaticoside from Centella asiatica(Umbelliferae). Crystallization was from dioxane (Mahato et al., 1987).X-ray diffraction analysis was also successful for confirmation of thestructure of mollic acid 3-β-D-glucoside (Pegel and Rogers, 1985).

X-ray crystallography is especially useful in solving structuralproblems of aglycones. Useful information for the determination of thestructure of the aglycone of alatoside A from Sesamum alatum(Pedaliaceae) was obtained by an X-ray diffraction analysis of thecrystalline triacetate of the artifact produced after acid hydrolysis(Potterat et al., 1992). An X-ray crystallographic study of medicagenicacid the parent aglycone of medicagenic acid 3-O-glucoside from thetubers of Dolichos kilimandscharicus (Leguminosae), showed the moleculeto have cis-fused D and E rings. Ring C had a slightly distorted sofaconformation, while rings A, B, D and E had chair conformations(Stoeckti-Evans, 1989).

(vii) Cleavage Reactions

Triterpene saponins are glycosides in which the hemiacetal hydroxylgroups of saccharides in their cyclic pyranose or furanose forms buildacetals with a triterpene or steroid residue. The ether linkage betweenthe hemiacetal hydroxyl and the triterpene or steroid is known as aglycosidic linkage. The monosaccharide constituents of theoligosaccharides also are bound by ether linkages (interglycosidicbonds).

On complete hydrolysis of a glycoside, the glycoside linkage is cleavedto liberate the component monosaccharides and the non-carbohydratemoiety (the aglycone or genin). The non-carbohydrate portion from thehydrolysis of saponins is termed a sapogenol or sapogenin. All knownsaponins are O-glycosides, with ether or ester linkages.

Numerous chemical reactions and methods have been employed for breakingdown saponins into smaller units for more ready analysis (see, forexample, Kitagawa, 1981). Such methods will find particular use instructural determinations of triterpene saponins.

1. Acidic Hydrolysis

Acidic hydrolysis maybe carried out by refluxing the saponin in acid fora fixed length of time, for example, 4 h in 2-4 M hydrochloric acid. Theaqueous solution remaining after hydrolysis is extracted with diethylether, chloroform or ethyl acetate to obtain the aglycone. Extraction ofthe sugars from the aqueous layer is performed with pyridine, afterneutralizing the solution (with alkali or basic ion exchange resin)(Tschesche and Forstmann, 1957; Sandberg and Michel, 1962) andevaporation to dryness. The saponins are completely cleaved into theirconstituents by this method so information is obtained as to theidentity of the aglycone and the number and nature of monosaccharidespresent. If a prosapogenin (obtained after cleavage of an ester linkageby basic hydrolysis) is acid hydrolyzed, the nature of the sugar chainswhich are ether-linked to the aglycone can be established. An aqueousreaction medium can be replaced by alcohol or dioxane.

In addition to hydrochloric acid, sulfuric acid also maybe employed forthe hydrolysis of saponins. With sulfuric acid there is less chance ofdegradation or rearrangement of the molecule but cleavage of etherlinkages is not as efficient. A convenient method of obtaininggypsogenic acid from Dianthus saponins, for example, involved hydrolysiswith 1 M sulfuric acid in dioxane (Oshima et al., 1984). A comparativestudy of hydrolytic conditions with hydrochloric acid and sulfuric acidin water and water-ethanol has shown that the best recoveries ofsaccharides are achieved by heating the saponin for 2 h with 5% sulfuricacid/water in a sealed vacuum ampoule (Kikuchi et al., 1987). Somewhatmilder hydrolyses can be achieved with trifluoroacetic acid, forexample, by refluxing for 3 h in 1 M trifluoroacetic acid.

An alternative to the hydrolysis of saponins in solution is to hydrolyzethem directly on a TLC plate by treatment with hydrochloric acid vapors.Once the acid has been evaporated, normal elution with the TLC solventis performed in order to identify the monosaccharides present (Kartnigand Wegschaider, 1971; He, 1987). By this means, the terminal sugarsxylose and galactose were identified after partial hydrolysis ofagaveside B. The TLC plate was developed with the solventchloroform-methanol-water (8:5:1) and the detection was by means ofaniline-diphenylamine-H₃PO₄-methanol (1:1:5:48) (Uniyal et al., 1990).

2. Basic Hydrolysis

Cleavage of O-acylglycosidic sugar chains is achieved under basichydrolysis conditions, typically by refluxing with 0.5 M potassiumhydroxide. Alternatively, 1-20% ethanolic or methanolic solutions ofpotassium hydroxide may be used but there is a risk of methylation,especially of the carboxyl group of triterpene acids. Ion exchangerssuch as Dowex 1 provide mildly basic hydrolysis conditions (Bukharov andKarlin, 1970). Another method is to use lithium iodide in collidine(Kochetkov et al., 1964).

By carefully controlling the reaction conditions, it is possible toselectively cleave different ester moieties. For example, hydrolysis ofkizuta saponin K₁₁ by refluxing in 0.5 M potassium hydroxide for 30 minremoved the sugar at C-28 of the bidesmoside. However, stirring thesaponin for 20 h in 0.1 M potassium hydroxide at room temperatureselectively removed the acetate group on the C-28 ester glycosidic chain(Kizu et al., 1985b).

3. Partial Hydrolysis

In certain instances, when saponins have highly branched or long sugarchains, a procedure involving partial hydrolysis is necessary in orderto obtain fragments more accessible to structure elucidation. This canbe achieved with acid or, indeed, with enzymes. The oligosaccharideand/or the remaining saponin portions are isolated and thencharacterized.

For example, saponin from Phytolacca dodecandra (Phytolaccaceae) washydrolyzed by 0.1 M hydrochloric acid for 45 min, to give a mixture ofthree products. These compounds were separated by RP-LPLC and theirsugar sequences determined by MS, ¹³C-NMR and GC-MS of alditol acetates.Putting all this information together enabled the assignment of achemical formula for the compound, an oleanolic acid derivative (Dorsazand Hostettmann, 1986).

Hydrolysis in dioxane gives milder conditions and partial hydrolysis ispossible. In this example, the saponin was refluxed for 6 h indioxane-0.1 M hydrochloric acid (1:3) (Ikram et al., 1981). Anothermethod for partially. hydrolyzing saponins is to treat a solution of thetriterpene glycoside in alcohol with an alkali metal (sodium orpotassium) and then add a trace of water (Ogihara and Nose, 1986).

4. Hydrothermolysis

Hydrothermolysis of triterpene glycosides leads to the formation of thecorresponding aglycones and thus can aid structure determination. Themethod involves heating the glycoside with water or water-dioxane at100° C. to 140° C. for a period of 10 to 140 h, depending on the sample.For example, hydrothermolysis of the triterpene 3,28-O-bisglycosidesgives the corresponding 3-O-glycosides (Kim et al., 1992).

5. Enzymatic Hydrolysis

A very efficient and mild method for the cleavage of sugar residues fromsaponins without artifact formation is enzymatic hydrolysis. Althoughthe relevant hydrolases for all the sugars are not commerciallyavailable, cleavages of β-glucose residues by β-glucosidase areperfectly straightforward. A supplementary benefit of cleavage byspecific enzymes is that the anomeric configuration of the sugar moietyis automatically proved. Certain enzyme preparations which areparticularly contemplated for use in hydrolysis of triterpene glycosidesare β-galactosidase hydrolyses, cellulase, crude hesperidinase,pectinase, and naringinase.

A systematic study involving crude preparations of hesperidinase,naringinase, pectinase, cellulase, amylase and emulsin has shown thathesperidinase, naringinase and pectinase were the most effective inhydrolyzing ginsenosides (Kohda and Tanaka, 1975).

(viii) Analysis of Aglycones After Hydrolysis

Once hydrolysis is complete, aglycones can be separated from thehydrolysate either by simple filtration or by a water-organic solventpartition and analyzed against known triterpenes. The most common methodis by TLC, using a solvent such as diisopropyl ether-acetone (75:30).Spray reagents are frequently those employed for the analysis ofsaponins (see Table 2).

Gas-liquid chromatography requires derivatization of triterpenes. Forexample, methyl esters of oleanolic and ursolic acids have beenseparated by GC on a glass column packed with 30% 0V-17 or SE-30(Fokina, 1979). Triterpenes can be determined by GC after derivatizationwith N,O-bis(trimethylsilyl)acetamide and chlorotrimethylsilane, as isthe case for soyasapogenols A-E and medicagenic acid in alfalfa(Jurzysta and Jurzysta, 1978).

The technique of GC-MS also is valuable for the characterization ofsapogenins. The trimethylsilyl derivatives are normally prepared andthen analyzed in the spectrometer. An example is the application to theinvestigation of oleanane- and ursane-type triterpenes. Nine silylatedtriterpenes were separated by GC on OV-101 packing and their massspectral patterns were investigated; those containing a 12-en doublebond underwent a characteristic retro-Diels-Alder reaction(Burnouf-Radosevich et al., 1985). This technique has also been used forthe determination of triterpenes from licorice (Bombardelli et al.,1979).

HPLC analysis does not require derivatization and gives excellentreproducibility and sensitivity for the analysis of triterpenes. Bothnormal-phase (analysis of quinoa sapogenins; Burnouf-Radosevich andDelfel, 1984) and RP-HPLC (Lin et al., 1981) can be employed, but adisadvantage of RP-HPLC is that the compounds tend to precipitate in theaqueous mobile phases.

(ix) Analysis of Sugars After Hydrolysis

Analysis of the monosaccharides may be carried out by TLC on, forexample, silica gel plates with solvents such as ethyl acetatemethanol-water acetic acid (65:25:15:20) and n-butanol ethyl acetatei-propanol acetic acid water (35:100:60:35:30) (Shiraiwa et al., 1991).Detection is typically with p-anisidine phthalate, naphthoresorcin,thymolsulfuric acid (Kartnig and Wegschaider, 1971) ortriphenyltetrazolium chloride (Wallenfels, 1950; Kamel et al., 1991).Alternatively, a quantitative analysis of the monosaccharides ispossible by GC or HPLC.

A number of HPLC methods have been reported for analysis of sugarsincluding: analysis on NH₂-bonded columns with acetonitrile-water(75:25) (Glombitza and Kurth, 1987); analysis on C-18 columns(acetonitrile-water 4:1) with refractive index detection, forquantitative purposes, integration of the HPLC peaks was compared withstandards (Adinolfi et al., 1987); analysis on an Aminex ion exclusionHPX-87H column (BioRad) with 0.005 M sulfuric acid as eluent (0.4ml/min) (Adinolfi et al., 1990); and analysis of sugar p-bromobenzoates(formed by methanolysis of the saponin with 5% hydrochloricacid-methanol and subsequent p-bromobenzoylation of the methyl sugars)by HPLC and identification by comparison with authentic derivatives(Kawai et al., 1988; Sakamoto et al., 1992).

For GC, the persilylated sugars are used (Wulff, 1965) or a GC-MSanalysis of alditol acetate derivatives is carried out.GC-Fourier-transformed IR (FTIR) analysis of suitably derivatizedmonosaccharides is an alternative procedure (Chen and Snyder, 1989).

The most commonly found sugars are D-glucose, D-galactose, L-arabinose,D-xylose, D-fucose, L-rhamnose, D-quinovose, D-glucuronic acid andD-ribose.

IV. Derivatives of the Compounds of the Invention

As described in detail herein, it is contemplated that certain benefitsmay be achieved from the manipulation of the triterpene glycosides toprovide them with novel characteristics, a longer in vivo half-life orother beneficial properties. Such techniques include, but are notlimited to, manipulation or modification of the mixtures of triterpeneglycosides or an individual triterpene molecule itself, modification orremoval of sugars, and conjugation of triterpene compounds to inertcarriers, such as various protein or non-protein components, includingimmunoglobulins and Fc portions. It will be understood that longerhalf-life is not coextensive with the pharmaceutical compositions foruse in “slow release.” Slow release formulations are generally designedto give a constant drug level over an extended period. Increasing thehalf-life of a drug, such as a triterpene glycoside in accordance withthe present invention, is intended to result in high plasma levels uponadministration, which levels are maintained for a longer time, but whichlevels generally decay depending on the pharmacokinetics of thecompound.

(i) Conjugates of Triterpenes and Linked Molecules

As described above, the triterpene compounds of the invention identifiedherein may be linked to particular molecules in order to improve theefficacy of the triterpene glycosides in treating patients for anyailment treatable with the compounds of the invention. Illustrativeembodiment of such molecules include targeting agents and agents whichwill increase the in vivo half life of the triterpene compounds. Thetriterpene compounds may be linked to such secondary molecules in anyoperative manner that allows each region to perform its intendedfunction without significant impairment of biological activity, forexample, the anti-tumor activity of the compounds disclosed herein.

The triterpene compositions of the present invention may be directlylinked to a second compound or may be linked via a linking group. By theterm “linker group” is intended one or more bifunctional molecules whichcan be used to covalently couple the triterpene compounds or triterpenemixture to the agent and which do not interfere with the biologicalactivity of the triterpene compounds. The linker group may be attachedto any part of the triterpene so long as the point of attachment doesnot interfere with the biological activity, for example, the anti-tumoractivity of the compounds of the invention.

An exemplary embodiment for linking the triterpene compounds of theinvention to a second agent is by the preparation of an active ester ofthe triterpene followed by reaction of the active ester with anucleophilic functional group on the agent to be linked. The activeesters may be prepared, for example, by reaction of a carboxyl group onthe triterpene with an alcohol in the presence of a dehydration agentsuch as dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI). The useof EDC to form conjugates is disclosed in U.S. Pat. No. 4,526,714; PCTAppl. Publ. No. WO91/01750, and Arnon et al., 1980, the disclosures ofwhich are specifically incorporated herein by reference in theirentirety. The agent to be linked to the triterpene, for example, atumor-specific antibody, is then mixed with the activated ester inaqueous solution to give the conjugate.

Where a linker group between the triterpene and the agent is desired,the active ester of the triterpene glycoside may be prepared asdescribed above and reacted with a linker group, for example,2-aminoethanol, an alkylene diamine, an amino acid such as glycine, or acarboxy-protected amino acid such as glycine tert-butyl ester. If thelinker contains a protected carboxy group, the protecting group isremoved and the active ester of the linker is prepared (as describedabove). The active ester is then reacted with the second molecule togive the conjugate. Alternatively, the second agent could be derivatizedwith succinic anhydride to give an agent-succinate conjugate which maybe condensed in the presence of EDC or EDCI with a triterpene-linkerderivative having a free amino or hydroxyl group on the linker (see, forexample, WO91/01750, the disclosure of which is specificallyincorporated herein by reference in its entirety).

It also is possible to prepare a triterpene glycoside conjugatecomprising a linker with a free amino group and crosslink the free aminogroup with a heterobifunctional cross linker such as sulfosuccinimidyl4-(N-maleimidocyclohexane)-1-carboxylate which will react with the freesulfhydryl groups of protein antigens.

The triterpene glycoside also may be coupled to a linker group byreaction of the aldehyde group with an amino linker to form anintermediate imine conjugate, followed by reduction with sodiumborohydride or sodium cyanoborohydride. Examples of such linkers includeamino alcohols such as 2-aminoethanol and diamino compounds such asethylenediamine, 1,2-propylenediamine, 1,5-pentanediamine,1,6-hexanediamine, and the like. The triterpene glycoside may then becoupled to the linker by first forming the succinated derivative withsuccinic anhydride followed by condensation with the triterpeneglycoside-linker conjugate with DCC, EDC or EDCI.

In addition, the triterpene glycoside or aglycone may be oxidized withperiodate and the dialdehyde produced therefrom condensed with an aminoalcohol or diamino compound listed above. The free hydroxyl or aminogroup on the linker may then be condensed with the succinate derivativeof the antigen in the presence of DCC, EDC or EDCI. Many types oflinkers are known in the art and may be used in the creation oftriterpene conjugates. A list of exemplary linkers for use with theinvention is given below, in Table 4.

TABLE 4 Hetero-Bifunctional Cross-Linkers Spacer Arm Reactive Advantagesand Length\after Linker Toward Applications cross-linking SMPT Primaryamines Greater stability 11.2 A Sulfhydryls SPDP Primary aminesThiolation  6.8 A Sulfhydryls Cleavable cross-linking LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Sulfo-LC-SPDP Primaryamines Extended spacer arm 15.6 A Sulfhydryls Water-soluble SMCC Primaryamines Stable maleimide reactive 11.6 A Sulfhydryls groupEnzyme-antibody conjugation Hapten-carrier protein conjugationSulfo-SMCC Primary amines Stable maleimide reactive 11.6 A Sulfhydrylsgroup Water-soluble Enzyme-antibody conjugation MBS Primary aminesEnzyme-antibody  9.9 A Sulfhydryls conjugation Hapten-carrier proteinconjugation Sulfo-MBS Primary amines Water-soluble  9.9 A SulfhydrylsSIAB Primary amines Enzyme-antibody 10.6 A Sulfhydryls conjugationSulfo-SIAB Primary amines Water-soluble 10.6 A Sulfhydryls SMPB Primaryamines Extended spacer arm 14.5 A Sulfhydryls Enzyme-antibodyconjugation Sulfo-SMPB Primary amines Extended spacer arm 14.5 ASulfhydryls Water-soluble EDC/Sulfo-N Primary amines Hapten-Carrierconjugation  0 HS Carboxyl groups ABH Carbohydrates Reacts with sugargroups 11.9 A Nonselective

(ii) Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active compounds. By creating such analogs, it is possibleto fashion drugs which are more active or stable than the naturalmolecules, which have different susceptibility to alteration or whichmay affect the function of various other molecules. In one approach, onewould generate a three-dimensional structure for the triterpenecompounds of the invention or a fragment thereof. This could beaccomplished by X-ray crystallography, computer modeling or by acombination of both approaches. An alternative approach, involves therandom replacement of functional groups throughout the triterpenemolecule, and the resulting affect on function determined.

It also is possible to isolate a triterpene compound specific antibody,selected by a functional assay, and then solve its crystal structure. Inprinciple, this approach yields a pharmacore upon which subsequent drugdesign can be based. It is possible to bypass protein crystallographyaltogether by generating anti-idiotypic antibodies to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of anti-idiotype would be expected to be an analog ofthe original antigen. The anti-idiotype could then be used to identifyand isolate peptides from banks of chemically- or biologically-producedpeptides. Selected peptides would then serve as the pharmacore.Anti-idiotypes may be generated using the methods described herein forproducing antibodies, using an antibody as the antigen.

Thus, one may design drugs which have improved biological activity, forexample, anti-tumor activity, relative to a starting triterpenecompound. By virtue of the chemical isolation procedures anddescriptions herein, sufficient amounts of the triterpene compounds ofthe invention can be produced to perform crystallographic studies. Inaddition, knowledge of the chemical characteristics of these compoundspermits computer employed predictions of structure-functionrelationships.

V. Treatment of Cancer with the Triterpene Compounds of the Invention

In the development of cancer, mammalian cells go through a series ofgenetically determined changes that lead to abnormal proliferation. Thiscan occur in steps, generally referred to as (1) initiation: when anexternal agent or stimulus triggers a genetic change in one or morecells and (2) promotion: involving further genetic and metabolicchanges, which can include inflamation. During the “promotion stage,”cells begin a metabolic transition to a stage of cellular growth inwhich apoptosis is blocked.

Cancer cells are characterized by a loss of apoptotic control inaddition to a loss of control of the regulatory steps of the cell cycle.Cancer cells (malignant cells) escape normal growth control mechanismsthrough a series of metabolic changes during the initiation andpromotion stages at the onset of malignancy. These changes are aconsequence of genetic alterations in the cells. These geneticalterations may include (i) activating mutations and/or increasedexpression of protooncogenes and/or (ii) inactivating mutations and/ordecreased expression of one or more tumor suppressor genes. Mostoncogene and tumor suppressor gene products are components of signaltransduction pathways that control cell cycle entry or exit, promotedifferentiation, sense DNA damage and initiate repair mechanisms, and/orregulate cell death programs. Nearly all tumors have mutations inmultiple oncogenes and tumor suppressor genes. One can conclude thatcells employ multiple parallel mechanisms to regulate cell growth,differentiation, DNA damage control, and apoptosis.

The triterpene compounds of the invention can be administered to asubject in need thereof to treat the subject either prophylacticallypreventing cancer or therapeutically after the detection of cancer. Toinhibit the initiation and promotion of cancer, to kill cancer/malignantcells, to inhibit cell growth, to induce apoptosis, to inhibitmetastasis, to decrease tumor size and to otherwise reverse or reducethe malignant phenotype of tumor cells, using the methods andcompositions of the present invention, one would generally contact a“target” cell with the triterpene compositions described herein. Thismay be achieved by contacting a tumor or tumor cell with a singlecomposition or pharmacological formulation that includes the triterpenecompounds of the invention, or by contacting a tumor or tumor cell withmore than one distinct composition or formulation, at the same time,wherein one composition includes a triterpene of the invention and theother includes a second agent.

Preferred cancer cells for treatment with the instant invention includeepithelial cancers such as skin, colon, uterine, ovarian, pancreatic,lung, bladder, breast, renal and prostate tumor cells. Other targetcancer cells include cancers of the brain, liver, stomach, esophagus,head and neck, testicles, cervix, lymphatic system, larynx, esophagus,parotid, biliary tract, rectum, uterus, endometrium, kidney, bladder,and thyroid; including squamous cell carcinomas, adenocarcinomas, smallcell carcinomas, gliomas, neuroblastomas, and the like. However, thislist is for illustrative purposes only, and is not limiting, aspotentially any tumor cell could be treated with the triterpenecompounds of the instant invention. Assay methods for ascertaining therelative efficacy of the compounds of the invention in treating theabove types of tumor cells and other tumor cells are specificallydisclosed herein and will be apparent to those of skill in the art inlight of the present disclosure.

The compounds of the present invention are preferably administered as anutraceutical composition or a pharmaceutical composition comprising apharmaceutically or pharmacologically acceptable diluent or carrier. Thenature of the carrier is dependent on the chemical properties of thecompound(s) employed, including solubility properties, and/or the modeof administration. For example, if oral administration is desired, asolid carrier may be selected, and for i.v. administration a liquid saltsolution carrier may be used.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

a. Nutraceuticals

Nutraceutical compositions are preparations of natural ingredients thatare multi-component systems consisting of preferably synergistic naturalproducts and supplements to promote good health. Nutraceutical compoundscan be derived from medicinal plants. Information about numerous plantsand herbs used to prepare nutraceutical compositions has been compiledand is available in publications including the German Commission EMonographs, Botanical Safety Handbook, and HerbalGram, a quarterlypublication of the American Botanical Council which references numerousclinical trials that have been performed using nutraceuticals.

Information on description and constituents, modern uses, dosage (in avariety of forms), actions, contraindications, side effects,interactions with conventional drugs, mode of administration, durationof application, regulatory status, AHPA botanical safety rating, andcomments are available for a number of plants and include among othersbilberry, cascara, cat's claw, cayenne, cranberry, devil's claw, dongquai, echinacea, evening primrose oil, feverfew, garlic, ginger, ginkgo,Asian ginseng, Siberian ginseng, goldenseal, gotu kola, grape seed,green tea, hawthorn, kava, licorice, milk thistle, saw palmetto, St.John's wort, and valerian.

The actions of these nutraceutical compounds may be fast or/andshort-term or may help achieve long-term health objectives. The currentinvention focuses on phytomedicines derived from Acacia victoriae. Theinvention envisions nutraceutical compositions comprising dried andground Acacia victoriae roots and pods or extracts from these tissues ina pharmacologically acceptable medium as a natural approach for, amongother things, the prevention and treatment of cancer. The nutraceuticalcomposition may be used to prevent the initiation and promotion ofcarcinogenesis and also for the induction of apoptosis in malignantcancer cells. The nutraceutical compositions disclosed herein may alsobe used as anti-inflammatory, anti-fungicidal, anti-viral,anti-mutagenic, spermicidal or contraceptive, cardiovascular andcholesterol metabolism regulatory agents. The nutraceutical compositionsmay be contained in a medium such as a buffer, a solvent, a diluent, aninert carrier, an oil, a creme, or an edible material.

The nutraceutical may be orally administered and may be in the form of atablet or a capsule. Oral intake may be preferred for the treatment ofcolon cancer and other internal tumors.

Alternatively the nutraceutical may be in the form of an ointment whichhas extracts of Acacia victoriae roots or pods in an oil or cream whichcan be topically applied to the skin. This form of nutraceuticalcomposition is useful for the preventing the initiation of skin cancers.The use of these nutraceuticals formulations provide a method ofinhibiting the initiation and promotion of mammalian epithelial cells toa premalignant or malignant state wherein a therapeutically effectiveamount of the nutraceutical composition is administered to a givenmammalian cell. This is especially useful for epithelial cell cancerssuch as skin cancer.

b. Pharmaceuticals

The invention further describes isolated compositions from Acaciavictoriae which have been partially or wholly purified and structurallycharacterized. The purification and characterization of these triterpeneglycoside compounds is described in detail the Examples. D1, G1 and B1are three compositions that have been wholly purified and theirstructural characterization is almost complete (FIG. 39, FIG. 40 andFIG. 41). Bioassays performed with these compounds on cancer cell lineshas demonstrated cell growth inhibition and the induction of apoptosisin malignant cells (FIG. 43, FIG. 44). Furthermore, partially purifiedcompositions of these saponins isolated from Acacia victoriae alsodemonstrate chemoprotective effects in mice exposed to the carcinogenDMBA (FIGS. 8, 9, 11, 12 and 13). Thus, these compositions haveanti-cancer activities and work by several mechanisms to induceapoptosis in cancer cells. Pharmaceutical compositions of thesecompounds are envisioned as powerful chemotherapeutic drugs which may beused by themselves or in combination with other forms of cancer therapysuch as chemotherapy, radiation therapy, surgery, gene therapy andimmunotherapy. The combination therapies are described below in detail.One of skill in the art will determine the effective dosages and thecombination therapy regimen.

c. Methods of Administration

(i) Parenteral Administration

One embodiment of the invention provides formulations for parenteraladministration of triterpene compositions, e.g., formulated forinjection via the intravenous, intramuscular, sub-cutaneous or othersuch routes, including direct instillation into a tumor or disease site.The preparation of an aqueous composition that contains a triterpenecomposition will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection also can be prepared; and thepreparations also can be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

The triterpene compounds can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) whichare formed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups alsocan be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

(ii) Other Modes of Administration

Other modes of administration will also find use with the subjectinvention. For instance, the triterpene compounds of the invention maybe formulated in suppositories and, in some cases, aerosol andintranasal compositions. For suppositories, the vehicle composition willinclude traditional binders and carriers such as polyalkylene glycols ortriglycerides. Such suppositories may be formed from mixtures containingthe active ingredient in the range of about 0.5% to about 10% (w/w),preferably about 1% to about 2%.

Oral compositions may be prepared in the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders.These compositions can be administered, for example, by swallowing orinhaling. Where a pharmaceutical composition is to be inhaled, thecomposition will preferably comprise an aerosol. Exemplary proceduresfor the preparation of aqueous aerosols for use with the currentinvention may be found in U.S. Pat. No. 5,049,388, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.Preparation of dry aerosol preparations are described in, for example,U.S. Pat. No. 5,607,915, the disclosure of which is specificallyincorporated herein by reference in its entirety.

Also useful is the administration of the invention compounds directly intransdermal formulations with permeation enhancers such as DMSO. Thesecompositions can similarly include any other suitable carriers,excipients or deluents. Other topical formulations can be administeredto treat certain disease indications. For example, intranasalformulations may be prepared which include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations also may contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject compounds by the nasal mucosa.

(iii) Formulations and Treatments

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulation of choice can be accomplished using a varietyof excipients including, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin cellulose,magnesium carbonate, and the like.

Typically, the compounds of the instant invention will contain from lessthan 1% to about 95% of the active ingredient, preferably about 10% toabout 50%. Preferably, between about 10 mg/kg patient body weight perday and about 25 mg/kg patient body weight per day will be administeredto a patient. The frequency of administration will be determined by thecare given based on patient responsiveness. Other effective dosages canbe readily determined by one of ordinary skill in the art throughroutine trials establishing dose response curves.

Regardless of the mode of administration, suitable pharmaceuticalcompositions in accordance with the invention will generally include anamount of the triterpene composition admixed with an acceptablepharmaceutical diluent or excipient, such as a sterile aqueous solution,to give a range of final concentrations, depending on the intended use.The techniques of preparation are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, which reference is specifically incorporatedherein by reference in its entirety. It should be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

The therapeutically effective doses are readily determinable using ananimal model, as shown in the studies detailed herein. For example,experimental animals bearing solid tumors are frequently used tooptimize appropriate therapeutic doses prior to translating to aclinical environment. Such models are known to be very reliable inpredicting effective anti-cancer strategies.

In certain embodiments, it may be desirable to provide a continuoussupply of therapeutic compositions to the patient. For intravenous orintraarterial routes, this is accomplished by drip system. For topicalapplications, repeated application would be employed. For variousapproaches, delayed release formulations could be used that providedlimited but constant amounts of the therapeutic agent over and extendedperiod of time. For internal application, continuous perfusion of theregion of interest may be preferred. This could be accomplished bycatheterization, post-operatively in some cases, followed by continuousadministration of the therapeutic agent. The time period for perfusionwould be selected by the clinician for the particular patient andsituation, but times could range from about 1-2 hours, to 2-6 hours, toabout 6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2weeks or longer. Generally, the dose of the therapeutic composition viacontinuous perfusion will be equivalent to that given by single ormultiple injections, adjusted for the period of time over which theinjections are administered. It is believed that higher doses may beachieved via perfusion, however.

1. Treatment Protocol

Two primary approaches are envisioned by the inventors for the use ofthe triterpene compounds of the invention either alone or in combinationtherapy. The first is the use in metastatic cancer either in patientswho have not received prior chemo, radio, or biological therapy or inpreviously untreated patients. Patients would be treated by systemicadministration, that is, intravenous, subcutaneous, oral administrationor by intratumoral injection. The pharmaceutical dose(s) administeredwould preferably contain between 10 and 25 mg of the triterpenecompositions of the invention per kg of patient body weight per day,including about 13, 16, 19, and 22 mg/kg/day. Alternatively, the patientcould be treated with one or more pharmaceutical compositions comprisingfrom about 1 mg/kg/day of the triterpene compositions of the inventionto about 100 mg/kg/day, including about 3, 6, 9, 12, 15, 18, 21, 28, 30,40, 50, 60, 70, 80 and 90 mg/kg/day of the triterpene compositions ofthe invention.

The treatment course typically consists of daily treatment for a minimumof eight weeks or one injection weekly for a minimum of eight weeks.Upon election by the clinician, the regimen may be continued on the sameschedule until the tumor progresses or the lack of response is observed.

Another application of the compounds of the invention is in treatingpatients who have been rendered free of clinical disease by surgery,chemotherapy, and/or radiotherapy. Adjuvant therapy would beadministered in the same regimen as described above for a minimum of oneyear to prevent recurrent disease.

2. Prevention of Cancer with the Compounds of the Invention

Another application of the compounds and mixtures of the invention is inthe prevention of cancer in high risk groups. Such patients (forexample, those with genetically defined predisposition to tumors such asbreast cancer, colon cancer, skin cancer, and others) would be treatedby mouth (gastrointestinal tumors), topically on the skin (cutaneous),or by systemic administration for a minimum period of one year andperhaps longer to determine prevention of cancer. This use would includepatients and well defined pre-neoplastic lesions, such as colorectalpolyps or other premalignant lesions of the skin, breast, lung, or otherorgans.

3. Clinical Protocol

A clinical protocol has been designed by the inventors to facilitate thetreatment of cancer using the triterpene compounds of the invention. Inaccordance with this protocol, patients having histologic proof ofcancer, for example, ovarian cancer, pancreatic cancer, renal cancer,prostate cancer, lung, or bladder will be selected. Patients may, butneed not have received previous chemo-, radio- or gene therapies.Optimally, patients will have adequate bone marrow function (defined asperipheral absolute granulocyte count of >2,000/mm³ and platelet countof 100,000/mm³), adequate liver function (bilirubin≦1.5 mg/dl) andadequate renal function (creatinine<1.5 mg/dl).

The protocol calls for single dose administration, via intratumoralinjection, of a pharmaceutical composition containing about 10 to 25 mgof the triterpene compounds of the invention per kg of patient bodyweight. For tumors of ≧4 cm, the volume administered will be 4-10 ml(preferably 10 ml), while for tumors<4 cm, a volume of 1-3 ml will beused (preferably 3 ml). Multiple injections will be delivered for asingle dose, in 0.1-0.5 ml volumes, with spacing of approximately 1 cmor more.

The treatment course consists of about six doses, delivered over twoweeks. Upon election by the clinician, the regimen may be continued, sixdoses each two weeks, or on a less frequent (monthly, bimonthly,quarterly, etc.) basis.

Where patients are eligible for surgical resection, the tumor will betreated as described above for at least two consecutive two-weektreatment courses. Within one week of completion of the second (or more,e.g., third, fourth, fifth, sixth, seventh, eighth, etc.) course, thepatient will receive surgical resection. Prior to close of the incision,10 ml of a pharmaceutical composition containing the triterpenecompounds of the invention will be delivered to the surgical site(operative bed) and allowed to remain in contact for at least 60minutes. The wound is closed and a drain or catheter placed therein. Onthe third post-operative day, an additional 10 ml of the pharmaceuticalcomposition is administered via the drain and allowed to remain incontact with the operative bed for at least two hours. Removal bysuction is then performed, and the drain removed at a clinicallyappropriate time.

4. Treatment of Artificial and Natural Body Cavities

One of the prime sources of recurrent cancer is the residual,microscopic disease that remains at the primary tumor site, as well aslocally and regionally, following tumor excision. In addition, there areanalogous situations where natural body cavities are seeded bymicroscopic tumor cells. The effective treatment of such microscopicdisease would present a significant advance in therapeutic regimens.

Thus, in certain embodiments, a cancer may be removed by surgicalexcision, creating a “cavity.” Both at the time of surgery, andthereafter (periodically or continuously), the therapeutic compositionof the present invention is administered to the body cavity. This is, inessence, a “topical” treatment of the surface of the cavity. The volumeof the composition should be sufficient to ensure that the entiresurface of the cavity is contacted by the expression construct.

In one embodiment, administration simply will entail injection of thetherapeutic composition into the cavity formed by the tumor excision. Inanother embodiment, mechanical application via a sponge, swab or otherdevice may be desired. Either of these approaches can be used subsequentto the tumor removal as well as during the initial surgery. In stillanother embodiment, a catheter is inserted into the cavity prior toclosure of the surgical entry site. The cavity may then be continuouslyperfused for a desired period of time.

In another form of this treatment, the “topical” application of thetherapeutic composition is targeted at a natural body cavity such as themouth, pharynx, esophagus, larynx, trachea, pleural cavity, peritonealcavity, or hollow organ cavities including the bladder, colon or othervisceral organs. In this situation, there may or may not be asignificant, primary tumor in the cavity. The treatment targetsmicroscopic disease in the cavity, but incidentally may also affect aprimary tumor mass if it has not been previously removed or apre-neoplastic lesion which may be present within this cavity. Again, avariety of methods may be employed to affect the “topical” applicationinto these visceral organs or cavity surfaces. For example, the oralcavity in the pharynx may be affected by simply oral swishing andgargling with solutions. However, topical treatment within the larynxand trachea may require endoscopic visualization and topical delivery ofthe therapeutic composition. Visceral organs such as the bladder orcolonic mucosa may require indwelling catheters with infusion or againdirect visualization with a cystoscope or other endoscopic instrument.Cavities such as the pleural and peritoneal cavities may be accessed byindwelling catheters or surgical approaches which provide access tothose areas.

(iv) Therapeutic Kits

The present invention also provides therapeutic kits comprising thetriterpene compositions described herein. Such kits will generallycontain, in suitable container means, a pharmaceutically acceptableformulation of at least one triterpene compound in accordance with theinvention. The kits also may contain other pharmaceutically acceptableformulations, such as those containing components to target thetriterpene compound to distinct regions of a patient where treatment isneeded, or any one or more of a range of drugs which may work in concertwith the triterpene compounds, for example, chemotherapeutic agents.

The kits may have a single container means that contains the triterpenecompounds, with or without any additional components, or they may havedistinct container means for each desired agent. When the components ofthe kit are provided in one or more liquid solutions, the liquidsolution is an aqueous solution, with a sterile aqueous solution beingparticularly preferred. However, the components of the kit may beprovided as dried powder(s). When reagents or components are provided asa dry powder, the powder can be reconstituted by the addition of asuitable solvent. It is envisioned that the solvent also may be providedin another container means. The container means of the kit willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which the triterpene glycoside, and anyother desired agent, may be placed and, preferably, suitably aliquoted.Where additional components are included, the kit will also generallycontain a second vial or other container into which these are placed,enabling the administration of separated designed doses. The kits alsomay comprise a second/third container means for containing a sterile,pharmaceutically acceptable buffer or other diluent.

The kits also may contain a means by which to administer the triterpenecompositions to an animal or patient, e.g., one or more needles orsyringes, or even an eye dropper, pipette, or other such like apparatus,from which the formulation may be injected into the animal or applied toa diseased area of the body. The kits of the present invention will alsotypically include a means for containing the vials, or such like, andother component, in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials and other apparatus are placed and retained.

VI. Chemotherapeutic Combinations and Treatment

In certain embodiments of the present invention, it may be desirable toadminister the triterpene compositions of the invention in combinationwith one or more other agents having anti-tumor activity includingchemotherapeutics, radiation, and therapeutic proteins or genes. Thismay enhance the overall anti-tumor activity achieved by therapy with thecompounds of the invention alone, or may be used to prevent or combatmulti-drug tumor resistance.

To use the present invention in combination with the administration of asecond chemotherapeutic agent, one would simply administer to an animala triterpene composition in combination with the second chemotherapeuticagent in a manner effective to result in their combined anti-tumoractions within the animal. These agents would, therefore, be provided inan amount effective and for a period of time effective to result intheir combined presence within the tumor vasculature and their combinedactions in the tumor environment. To achieve this goal, the triterpenecomposition and chemotherapeutic agents may be administered to theanimal simultaneously, either in a single composition or as two distinctcompositions using different administration routes.

Alternatively, the triterpene composition treatment may precede orfollow the chemotherapeutic agent, radiation or protein or gene therapytreatment by intervals ranging from minutes to weeks. In embodimentswhere the second agent and triterpene composition are administeredseparately to the animal, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the additional agent and triterpene composition would still be ableto exert an advantageously combined effect on the tumor. In suchinstances, it is contemplated that one would contact the tumor with bothagents within about 5 minutes to about one week of each other and, morepreferably, within about 12-72 hours of each other, with a delay time ofonly about 24-48 hours being most preferred. In some situations, it maybe desirable to extend the time period for treatment significantly,where several days (2, 3, 4, 5, 6 or 7) or even several weeks (1, 2, 3,4, 5, 6, 7 or 8) lapse between the respective administrations. It alsois conceivable that more than one administration of either thetriterpene glycoside or the second agent will be desired. To achievetumor regression, both agents are delivered in a combined amounteffective to inhibit its growth, irrespective of the times foradministration.

A variety of agents are suitable for use in the combined treatmentmethods disclosed herein. Chemotherapeutic agents contemplated asexemplary include, e.g., etoposide (VP-16), adriamycin, 5-fluorouracil(5-FU), camptothecin, actinomycin-D, mitomycin C, and cisplatin (CDDP).

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating agents may be used. Cisplatin has been widely usedto treat cancer, with efficacious doses used in clinical applications of20 mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotideprecursors also may be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU is applicable in a widerange of carriers, including topical, with intravenous administration indoses ranging from 3 to 15 mg/kg/day being commonly used.

Exemplary chemotherapeutic agents that are useful in connection withcombined therapy are listed in Table 5. Each of the agents listedtherein are exemplary and by no means limiting. In this regard, theskilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition’, chapter 33, in particular pages 624-652. Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

TABLE 5 Chemotherapeutic Agents Useful In Neoplastic DiseaseNONPROPRIETARY NAMES CLASS TYPE OF AGENT (OTHER NAMES) DISEASEAlkylating Nitrogen Mustards Mechlorethamine (HN₂) Hodgkin's disease,non-Hodgkin's Agents lymphomas Cyclophosphamide Acute and chroniclymphocytic Ifosfamide leukemias, Hodgkin's disease, non-Hodgkin'slymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung, Wilms'tumor, cervix, testis, soft-tissue sarcomas Melphalan (L-sarcolysin)Multiple myeloma, breast, ovary Chlorambucil Chronic lymphocyticleukemia, primary macroglobulinemia, Hodgkin's disease, non-Hodgkin'slymphomas Ethylenimenes and Hexamethylmelamine Ovary MethylmelaminesThiotepa Bladder, breast, ovary Alkyl Sulfonates Busulfan Chronicgranulocytic leukemia Nitrosoureas Carmustine (BCNU) Hodgkin's disease,non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma,malignant melanoma Lomustine (CCNU) Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, small-cell lung Semustine Primary braintumors, stomach, colon (methyl-CCNU) Streptozocin Malignant pancreaticinsulinoma, (streptozotocin) malignant carcinoid Triazines Dacarbazine(DTIC; Malignant melanoma, Hodgkin'sdimethyltriazenoimidazolecarboxamide) disease, soft-tissue sarcomasAntimetabolites Folic Acid Analogs Methotrexate Acute lymphocyticleukemia, (amethopterin) choriocarcinoma, mycosis fungoides, breast,head and neck, lung, osteogenic sarcoma Pyrimidine Analogs Fluouracil(5-fluorouracil; Breast, colon, stomach, pancreas, 5-FU) ovary, head andneck, urinary Floxuridine bladder, premalignant skin lesions(fluorode-oxyuridine; (topical) FUdR) Cytarabine (cytosine Acutegranulocytic and acute arabinoside) lymphocytic leukemias Purine Analogsand Mercaptopurine Acute lymphocytic, acute Related Inhibitors(6-mercaptopurine; granulocytic and chronic granulocytic 6-MP) leukemiasThioguanine Acute granulocytic, acute (6-thioguanine; TG) lymphocyticand chronic granulocytic leukemias Pentostatin Hairy cell leukemia,mycosis (2-deoxycoformycin) fungoides, chronic lymphocytic leukemiaNatural Products Vinca Alkaloids Vinblastine (VLB) Hodgkin's disease,non-Hodgkin's lymphomas, breast, testis Vincristine Acute lymphocyticleukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin'sdisease, non-Hodgkin's lymphomas, small-cell lung EpipodophyllotoxinsEtoposide Testis, small-cell lung and other lung, Tertiposide breast,Hodgkin's disease, non-Hodgkin's lymphomas, acute granulocytic leukemia,Kaposi's sarcoma Antibiotics Dactinomycin Choriocarcinoma, Wilms' tumor,(actinomycin D) rhabdomyosarcoma, testis, Kaposi's sarcoma DaunorubicinAcute granulocytic and acute (daunomycin; lymphocytic leukemiasrubidomycin) Doxorubicin Soft-tissue, osteogenic and other sarcomas;Hodgkin's disease, non-Hodgkin's lymphomas, acute leukemias, breast,genitourinary, thyroid, lung, stomach, neuroblastoma Bleomycin Testis,head and neck, skin, esophagus, lung and genitourinary tract; Hodgkin'sdisease, non-Hodgkin's lymphomas Plicamycin (mithramycin) Testis,malignant hypercalcemia Mitomycin (mitomycin C) Stomach, cervix, colon,breast, pancreas, bladder, head and neck Enzymes L-Asparaginase Acutelymphocytic leukemia Biological Response Interferon alfa Hairy cellleukemia., Kaposi's Modifiers sarcoma, melanoma, carcinoid, renal cell,ovary, bladder, non-Hodgkin's lymphomas, mycosis fungoides, multiplemyeloma, chronic granulocytic leukemia Miscellaneous PlatinumCoordination Cisplatin (cis-DDP) Testis, ovary, bladder, head and AgentsComplexes Carboplatin neck, lung, thyroid, cervix, endometrium,neuroblastoma, osteogenic sarcoma Anthracenedione Mitoxantrone Acutegranulocytic leukemia, breast Substituted Urea Hydroxyurea Chronicgranulocytic leukemia, polycythemia vera, essental thrombocytosis,malignant melanoma Methyl Hydrazine Procarbazine Hodgkin's diseaseDerivative (N-methylhydrazine, MIH) Adrenocortical Mitotane (o,p′-DDD)Adrenal cortex Suppressant Aminoglutethimide Breast Hormones andAdrenocorticosteroids Prednisone (several other Acute and chroniclymphocytic Antagonists equivalent leukemias, non-Hodgkin's lymphomas,preparations available) Hodgkin's disease, breast ProgestinsHydroxyprogesterone Endometrium, breast caproate Medroxyprogesteroneacetate Megestrol acetate Estrogens Diethylstilbestrol Breast, prostateEthinyl estradiol (other preparations available) Antiestrogen TamoxifenBreast Androgens Testosterone propionate Breast Fluoxymesterone (otherpreparations available) Antiandrogen Flutamide ProstateGonadotropin-releasing Leuprolide Prostate hormone analog

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors also are contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

VII. Targeted Cancer Therapy

The triterpene compounds described herein may be linked to one or moremolecules which target the compounds to tumor cells. Targeting isbeneficial in that it can be used to increase the overall levels of adrug at the site of treatment, for example, at tumor sites, whileminimizing systemic exposure to the drug. In common with thechemotherapeutic agents discussed above, it is possible that the use ofa targeted triterpene compound may be used in combination with a secondagent, such as a chemotherapeutic agent. Both the triterpene and thesecond agent be directed to the same or different targets within thetumor environment. This should result in additive, greater than additiveor even markedly synergistic results.

Exemplary targeting agents employed in combination with the triterpenecompounds of the present invention will be those targeting agents thatare capable of delivering the triterpene molecules to the tumor region,i.e., capable of localizing within a tumor site. Similarly desired willbe those agents which target the vasculature of a tumor region. Thetargeting of the triterpene glycoside compounds is specificallycontemplated to allow for greater effective concentrations in tumorregions without or with the minimization of potential side effects whichcould be observed with a somewhat wider or systemic distribution of thetriterpene compounds. Specifically, the targeting agent may be directedto components of tumor cells; components of tumor vasculature;components that bind to, or are generally associated with, tumor cells;components that bind to, or are generally associated with, tumorvasculature; components of the tumor extracellular matrix or stroma orthose bound therein; and even cell types found within the tumorvasculature.

(i) Tumor Cell Targets and Antibodies

The malignant cells that make up the tumor may be targeted using abispecific antibody that has a region capable of binding to a relativelyspecific marker or antigen of the tumor cell. For example, specifictumor cell inhibition or killing may be achieved by the binding of anantibody-triterpene composition conjugate to a target tumor cell.

Many so-called “tumor antigens” have been described, any one which couldbe employed as a target in connection with the targeted aspects of thepresent invention. A large number of exemplary solid tumor-associatedantigens are listed herein below. The preparation and use of antibodiesagainst such antigens is well within the skill of the art andspecifically disclosed herein. Exemplary antibodies include those fromgynecological tumor sites (see, e.g., the ATCC Catalogue): OC 125; OC133; OMI; Mo v1; Mo v2; 3C2; 4C7; 113₃, DU-PAN-2; F 36/22; 4F₇/7A_(10;)OV-TL3; B72.3; DF_(3;) 2C₈/2F_(7;) MF 116; Mov18; CEA 11-H5; CA 19-9(1116NS 19-9); H17-E2; 791T/36; NDOG_(2;) 11317; 4D5, 3H4, 7C2, 6E9,2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; HMFG2; 3.14.A3; from breast tumorsites: DF3; NCRC-11; 3C6F9; MBE6; CLNH5; MAC 40/43; EMA; HMFG1 HFMG2;3.15.C3; M3, M8, M24; M18; 67-D-11; D547Sp, D75P3, H222; Anti-EGF; LR-3;TA1; H59; 10-3D-2; HmAB1,2; MBR 1,2,3; 24-17-1; 24-17-2 (3E1-2);F36/22.M7/105; C11, G3, H7; B6-2; B1-1; Cam 17-1; SM3; SM4; C-Mul (566);4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, 5B8; OC 125; MO v2;DU-PAN-2; 4F₇/7A_(10;) DF₃,-B72-3; cccccCEA 11; H17-E2; 3-14-A3; FO23C5;from colorectal tumor sites: B72-3; (17-1A) 1083-17-1A; CO17-1A;ZCE-025; AB2; HT-29-15; 250-30.6; 44X14; A7; GA73-3; 791T/36; 28A32;28.19.8; X MMCO-791; DU-PAN-2; ID_(3;) CEA 11-H5; 2C₈/2F_(7;) CA-19-9(1116NS 19-9); PR5C5; PR4D2; PR4D1; from melanoma sites 4-1; 8-2 M_(17;)96.5; 118-1, 133-2, (113-2); L₁, L₁₀, R₁₀(R₁₉); I_(12;) K_(5;) 6-1; R24;5-1; 225.28S; 465.12S; 9-2-27; F11; 376.96S; 465.12S; 15-75; 15.95;Mel-14; Mel-12; Me3-TB7; 225.28SD; 763.24TS; 705F6; 436910; M148; fromgastrointestinal tumors: ID3; DU-PAN-2; OV-TL3; B72-3; CEA 11-H5;3-14-A3; C COLI; CA-19-9 (1116NS 19-9) and CA50; OC125; from lungtumors: 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; MO v2;B72-3; DU-PAN-2; CEA 11-H5; MUC 8-22; MUC 2-63; MUC 2-39; MUC 7-39; andfrom miscellaneous tumors: PAb 240; PAb 246; PAb 1801; ERIC-1; M148;FMH25; 6.1; CAl; 3F8; 4F₇/7A_(10;) 2C₈/2F_(7;) CEA 11-H5.

Another means of defining and targeting a tumor is in terms of thecharacteristics of a tumor cell itself, rather than describing thebiochemical properties of an antigen expressed b_(y) the cell. A numberof exemplary tumor cell lines are known and may be used for thepreparation of targeting agents. For example, whole cells or cellhomogenates from known tumor lines could be used to prepare anti-tumorantibodies for the targeting of related tumors types. Similarly, suchtumor cell lines may find use in the implementation of various in vitroassays. In this regard, the skilled artisan is referred to the ATCCcatalogue for the purpose of exemplifying human tumor cell lines thatare publicly available (from ATCC Catalogue). Exemplary cell linesinclude J82; RT4; ScaBER; T24; TCCSUP; 5637; SK-N-MC; SK-N-SH; SW 1088;SW 1783; U-87 MG; U-118 MG; U-138 MG; U-373 MG; Y79; BT-20; BT-474;MCF7; MDA-MB-134-VI; MDA-MD-157; MDA-MB-175-VII; MDA-MB-361; SK-BR-3;C-33 A; HT-3; ME-180; MS751; SiHa; JEG-3; Caco-2; HT-29; SK-CO-1; HuTu80; A-253; FaDu; A-498; A-704; Caki-1; Caki-2; SK-NEP-1; SW 839;SK-HEP-1; A-427; Calu-1; Calu-3; Calu-6; SK-LU-1; SK-MES-1; SW 900; EB1;EB2; P3HR-1; HT-144; Malme-3M; RPMI-7951; SK-MEL-1; SK-MEL-2; SK-MEL-3;SK-MEL-5; SK-MEL-24; SK-MEL-28; SK-MEL-31; Caov-3; Caov-4; SK-OV-3; SW626; Capan-1; Capan-2; DU 145; A-204; Saos-2; SK-ES-1; SK-LMS-1; SW684;. SW 872; SW 982; SW 1353; U-2 OS; Malme-3; KATO III; Cate-1B;Tera-1; Tera-2; SW579; AN3 CA; HEC-1-A; HEC-1-B; SK-UT-1; SK-UT-1B; SW954; SW 962; NCI-H69; NCI-H128; BT-483; BT-549; DU4475; HBL-100; Hs578Bst; Hs 578T; MDA-MB-330; MDA-MB-415; MDA-MB-435S; MDA-MB-436;MDA-MB-453; MDA-MB-468; T-47D; Hs 766T; Hs 746T; Hs 695T; Hs 683; Hs294T; Hs 602; JAR; Hs 445; Hs 700T; 114; Hs 696; Hs 913T; Hs 729; FHs738Lu; FHs 173We; FHs 738B1; NIH:OVCAR-3 Hs 67; RD-ES; ChaGo K-1;WERI-Rb-1; NCI-11446; NCI-H209; NCI-H 146; NCI-H441; NCI-1182; H9;NCI-H460; NCI-H596; NCI-H676B; NCI-H345; NCI-H820; NCI-H520; NCI-H661;NCI-H510A; D283 Med; Daoy; D341 Med; AML-193 and MV4-11.

One may consult the ATCC Catalogue of any subsequent year to identifyother appropriate cell lines. Also, if a particular cell type isdesired, the means for obtaining such cells, and/or their instantlyavailable source, will be known to those of skill in the particular art.An analysis of the scientific literature will thus readily reveal anappropriate choice of cell for any tumor cell type desired to betargeted.

As explained above, antibodies constitute a straightforward means ofrecognizing a tumor antigen target. An extensive number of antibodiesare known that are directed against solid tumor antigens. Certain usefulanti-tumor antibodies are listed above. However, as will be known'tothose of skill in the art, certain of the antibodies listed will nothave the appropriate biochemical properties, or may not be of sufficienttumor specificity, to be of use therapeutically. An example is MUC8-22that recognizes a cytoplasmic antigen. Antibodies such as these willgenerally be of use only in investigational embodiments, such as inmodel systems or screening assays.

Generally speaking, antibodies for use in these aspects of the presentinvention will preferably recognize antigens that are accessible on thecell-surface and that are preferentially, or specifically, expressed bytumor cells. Such antibodies will also preferably exhibit properties ofhigh affinity, such as exhibiting a K_(d) of <200 nM, and preferably, of<100 nM, and will not show significant reactivity with life-sustainingnormal tissues, such as one or more tissues selected from heart, kidney,brain, liver, bone marrow, colon, breast, prostate, thyroid, gallbladder, lung, adrenals, muscle, nerve fibers, pancreas, skin, or otherlife-sustaining organ or tissue in the human body. The “life-sustaining”tissues that are the most important for the purposes of the presentinvention, from the standpoint of low reactivity, include heart, kidney,central and peripheral nervous system tissues and liver. The term“significant reactivity,” as used herein, refers to an antibody orantibody fragment that, when applied to the particular tissue underconditions suitable for immunohistochemistry, will elicit either nostaining or negligible staining with only a few positive cells scatteredamong a field of mostly negative cells.

Particularly promising antibodies contemplated for use in the presentinvention are those having high selectivity for the solid tumor. Forexample, antibodies binding to TAG 72 and the HER-2 proto-oncogeneprotein, which are selectively found on the surfaces of many breast,lung and colorectal cancers (Thor et al., 1986; Colcher et al., 1987;Shepard et al., 1991); MOv18 and OV-TL3 and antibodies that bind to themilk mucin core protein and human milk fat globule (Miotti et al., 1985;Burchell et al., 1983); and the antibody 9.2.27 that binds to the highM_(r) melanoma antigens (Reisfeld et al., 1982). Further usefulantibodies are those against the folate-binding protein, which is knownto be homogeneously expressed in almost all ovarian carcinomas; thoseagainst the erb family of oncogenes that are over-expressed in squamouscell carcinomas and the majority of gliomas; and other antibodies knownto be the subject of ongoing pre-clinical and clinical evaluation.

The antibodies B3, KSI/4, CC49, 260F9, XMMCO-791, D612 and SM3 arebelieved to be particularly suitable for use in clinical embodiments,following the standard pre-clinical testing routinely practiced in theart. B3 (U.S. Pat. No. 5,242,813; Brinkmann et al., 1991) has ATCCAccession No. HB 10573; KS1/4 can be made as described in U.S. Pat. No.4,975,369; and D612 (U.S. Pat. No. 5,183,756) has ATCC Accession No. HB9796.

Another means of defining a tumor-associated target is in terms of thecharacteristics of the tumor cell, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,the inventors contemplate that any antibody that preferentially binds toa tumor cell may be used as the targeting component of antriterpene-targeting conjugate. The preferential tumor cell binding isagain based upon the antibody exhibiting high affinity for the tumorcell and not having significant reactivity with life-sustaining normalcells or tissues, as defined above.

The invention also provides several means for generating an antibody foruse in the targeting of triterpene glycosides to tumor cells asdescribed herein. To generate a tumor cell-specific antibody, one wouldimmunize an animal with a composition comprising a tumor cell antigenand, as described more fully below, select a resultant antibody withappropriate specificity. The immunizing composition may contain apurified, or partially purified, preparation of any of the antigenslisted above; a composition, such as a membrane preparation, enrichedfor any of the antigens in listed above; any of the cells listed above;or a mixture or population of cells that include any of the cell typeslisted above.

Of course, regardless of the source of the antibody, in the practice ofthe invention in human treatment, one will prefer to ensure in advancethat the clinically-targeted tumor expresses the antigen ultimatelyselected. This is achieved by means of a fairly straightforward assayinvolving antigenically testing a tumor tissue sample, for example, asurgical biopsy, or perhaps testing for circulating shed antigen. Thiscan readily be carried out in an immunological screening assay such asan ELISA (enzyme-linked immunosorbent assay), wherein the bindingaffinity of antibodies from a “bank” of hybridomas are tested forreactivity against the tumor. Antibodies demonstrating appropriate tumorselectivity and affinity are then selected for the preparation ofbispecific antibodies of the present invention.

Due to the well-known phenomenon of cross-reactivity, it is contemplatedthat useful antibodies may result from immunization protocols in whichthe antigens originally employed were derived from an animal, such as amouse or a primate, in addition to those in which the original antigenswere obtained from a human cell. Where antigens of human origin areused, they may be obtained from a human tumor cell line, or may beprepared by obtaining a biological sample from a particular patient inquestion. Indeed, methods for the development of antibodies that are“custom-tailored” to the patient's tumor are known (Stevenson et al.,1990) and are contemplated for use in connection with this invention.

1. Methods for Antibody Production

As indicated, antibodies may find use in particular embodiments of theinstant invention. For example, antibodies may be produced which arespecific for a particular region in a patient or a particular tissuetype. These antibodies may then be conjugated to a triterpene compoundof the invention, thereby allowing the specific targeting of thetriterpene compounds to the tissue for which the antibody is directedto. An exemplary embodiment of such an antibody is one which binds to atumor cell. In a preferred embodiment of the invention, an antibody is amonoclonal antibody. Means for preparing and characterizing monoclonaland polyclonal antibodies are well known in the art and specificallydisclosed herein below (see, e.g., Howell and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising the desired target antigen and collectingantisera from that immunized animal. A wide range of animal species canbe used for the production of antisera. Typically an animal used forproduction of anti-antisera is a non-human animal including rabbits,mice, rats, hamsters, pigs or horses. Because of the relatively largeblood volume of rabbits, a rabbit is a preferred choice for productionof polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of particular cell types or,alternatively, the compounds of the present invention, can be used toimmunize one or more experimental animals, such as a rabbit or mouse,which will then proceed to produce specific antibodies against theantigens. Polyclonal antisera may be obtained, after allowing time forantibody generation, simply by bleeding the animal and preparing serumsamples from the whole blood.

It is believed that the monoclonal antibodies of the present inventionwill find useful application in immunochemical procedures which may beapplied to screening for the presence of the triterpene compounds of theinvention in species other than Acacia victoriae, or in other procedureswhich may utilize antibodies specific to particular antigens. Asdiscussed, an exemplary embodiment of the use of antibodies with theinvention comprises preparing antibodies directed to tumor-specificantigens, linking the antibodies to the triterpene compounds of theinvention, and treating human patients with the antigen-triterpeneconjugate, whereby the triterpene compounds of the invention arespecifically targeted to tumor cells or other cells which are involvedin a condition which can be treated with the triterpene compounds of theinvention. In general, both polyclonal and monoclonal antibodies againstvarious antigens may be employed in different embodiments of theinvention. For example, they may be employed in purifying triterpenecompounds in an antibody affinity column. Means for preparing andcharacterizing such antibodies are well known in the art and aredisclosed in, for example, Harlow and Lane, 1988, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin also canbe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of route's can be used to administerthe immunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection also may be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, the disclosure of whichis specifically incorporated herein by reference in its entirety.Typically, this technique involves immunizing a suitable animal with aselected immunogen composition, for example, a purified or partiallypurified tumor-specific antigen, polypeptide or peptide or tumor cell.The immunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep or frog cells alsois possible. The use of rats may provide certain advantages (Goding,1986), but mice are preferred, with the BALB/c mouse being mostpreferred as this is most routinely used and generally gives a higherpercentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art. For example, where the immunized animal is a mouse,one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one mayuse R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions(see, e.g., Goding, 1986; Campbell, 1984; and the ATCC Catalogue).

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et aL, (1977). The use of electricallyinduced fusion methods also is appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

(iii) Further Tumor Cell Targets and Binding Ligands

In addition to the use of antibodies, other ligands could be employed todirect a triterpene compounds of the invention to a tumor site bybinding to a tumor cell antigen. For tumor antigens that areover-expressed receptors (e.g., an estrogen receptor, EGF receptor), ormutant receptors, the corresponding ligands could be used as targetingagents.

In an analogous manner to endothelial cell receptor ligands, there maybe components that are specifically, or preferentially, bound to tumorcells. For example, if a tumor antigen is an over-expressed receptor,the tumor cell may be coated with a specific ligand in vivo. Therefore,the ligand could then be targeted either with an antibody against theligand, or with a form of the receptor itself. Specific examples ofthese type of targeting agents are antibodies against TIE-1 or TIE-2ligands, antibodies against platelet factor 4, and leukocyte adhesionbinding protein.

(iv) Toxins

For certain applications, it is envisioned that the second therapeuticagents used in combination with the triterpene compounds describedherein will be pharmacologic agents conjugated to antibodies or growthfactors, particularly cytotoxic or otherwise anti-cellular agents havingthe ability to kill or suppress the growth or cell division ofendothelial cells. In general, the invention contemplates the use of anypharmacologic agent, including and in supplement to the triterpenecompounds described herein, that can be conjugated to a targeting agent,preferably an antibody, and delivered in active form to the targeted,tumor cells. Exemplary anti-cellular agents include chemotherapeuticagents, radioisotopes as well as cytotoxins. In the case ofchemotherapeutic agents, the inventors believe that agents such as asteroid hormone; an anti-metabolite such as cytosine arabinoside,fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycinC; a vinca alkaloid; demecolcine; etoposide; mithramycin; or ananti-tumor alkylating agent such as chlorambucil or melphalan, will beparticularly preferred. Other embodiments may include agents such as acytokine, growth factor, bacterial endotoxin or the lipid A moiety ofbacterial endotoxin. In any event, it is believed that agents such asthese may, if desired, be successfully linked together with thetriterpene compounds of the invention to targeting agents, preferably anantibody, in a manner that will allow their targeting, internalization,release or presentation to blood components at the site of the targetedcells as required using known conjugation technology (see, e.g., Ghoseet al., 1983 and Ghose et al., 1987).

A variety of chemotherapeutic and other pharmacologic agents have nowbeen successfully conjugated to antibodies and shown to functionpharmacologically (see, e.g., Vaickus et al., 1991). Exemplaryantineoplastic agents that have been investigated include doxorubicin,daunomycin, methotrexate, vinblastine, and various others (Dillman etal., 1988; Pietersz et al, 1988). Moreover, the attachment of otheragents such as neocarzinostatin (Kimura et al., 1983), macromycin(Manabe et al., 1984), trenimon (Ghose, 1982) and α-amanitin (Davis &Preston, 1981) has been described. Specific means for preparingconjugates between the triterpene compounds of the instant invention andappropriate targeting molecules are specifically disclosed herein above.

VIII. Other Uses of the Compounds of the Invention

The inventors specifically contemplate the use of the compounds of thisinvention for a range of applications in addition to the treatment orprevention of cancer. In particular, the inventors contemplate the useof the triterpene compounds of the invention as solvents, anti-fungaland anti-viral agents, piscicides or molluscicides, contraceptives,antihelmintics, UV-protectants, expectorants, diuretics,anti-inflammatory agents, regulators of cholesterol metabolism,cardiovascular effectors, anti-ulcer agents, analgesics, sedatives,immunomodulators, antipyretics, angiogenesis regulators, as agents fordecreasing capillary fragility, as agents to combat the effects ofaging, and as agents for improving cognition and memory.

The compounds of this invention have a role in the regulation ofangiogenesis. Angiogenesis or neovascularization is defined as thegrowth of new blood vessels. Tumors and cancers induce angiogenesis toprovide a life-line for oxygen and nutrients for the tumor to thrive.The development of new blood vessels also provide exits for malignantcancer cells to spread to other parts of the body. Angiogenesisinhibition therefore benefits cancer patients. On the other hand,angiogenesis is required at times such as wound healing. These woundscan be external wounds or internal organ wounds that result fromaccidents, burns, injury and surgery. Thus, agents that promoteangiogenesis have a great potential for use in therapy for woundhealing.

The application of the compounds of the invention for modulation ofcholesterol metabolism is also contemplated. In particular, thecompounds and nutraceuticals of the invention are contemplated for usein lowering the serum cholesterol levels of human patients. Therefore,by treating patients with the triterpene compounds of the invention,either orally or intravenously, it is believed the morbidity associatedwith high cholesterol and related cardiovascular diseases may bedecreased.

For the treatment of cardiovascular conditions, it is contemplated thatthe compounds of the invention may be used for the treatment ofarrhythmic action and further may be used as a vascular relaxant,resulting in an antihypertensive activity.

Another particularly significant use contemplated for the compounds ofthe invention is as an anti-inflammatory agent. The inventors have shownthat the active triterpene compounds of the invention are potentinhibitors of transcription factor NF-κB, which plays an important rolein the inflammatory response. This finding is particularly significantgiven the increasing amount of evidence suggesting the central role ofinflammatory response in carcinogenesis. Treatment of patients with thetriterpene compounds provided herein may, therefore, potentiallyalleviate a wide degree of ailments associated with inflammation,including tumorigenesis and tissue damage.

The initial stages of an inflammatory response are characterized byincreased blood vessel permeability and release (exudation) ofhistamine, serotonin and basic polypeptides and proteins. This isaccompanied by hyperaemia and oedema formation. Subsequently, there iscellular infiltration and formation of new conjunctive tissue. It isbelieved that treatment with the compounds of the invention can limitthese early stages of inflammation and, thereby, decrease the negativeeffects associated with the inflammatory condition.

The plant species from which the compounds of the invention wereidentified, Acacia victoriae, was selected, in part, because it isnative to and regions. An important function of the metabolism of plantsfrom these regions is the production of compounds which protect cellsfrom ultraviolet radiation. The inventors specifically contemplate thatthe triterpene compounds of the invention are capable of serving as suchUV-protectants. It is, therefore, believed that the compounds of theinvention will find wide use in applications in which protection fromultraviolet radiation is desired. For example, a suitable applicationcomprises the use of the triterpene compounds of the invention as aningredient in sunblock, or other similar lotions for application tohuman skin.

The potential benefit of such a composition is indicated by thechemoprotective effects demonstrated for the compounds of the inventionherein. Lotions and sunblocks containing the triterpene compounds of theinvention would, therefore, be particularly suited to those with apredisposition to various forms of skin cancer. Examples of such includethe fair skinned and, particularly, those with a genetic predispositionto skin cancel. Such predispositions include heritable oncogenemutations or mutations in the cellular mechanisms which mediate DNArepair to UV-induced damage. Particularly significant are mutations ingenes controlling genetic repair mechanisms, for example, the excisionof UV-induced thymine-thymine dimers. Similarly, the compounds of theinvention could be added to any other composition for which increasedUV-protection is desired, and these compounds applied to any animate orinanimate object for which UV-protection is sought.

Other possible application of the triterpene compounds includeprotection in the central nervous system damage, in effect, memory lossor enhanced cognitive function, use as an antioxidant (monitoring bloodlevels of oxidative molecules), or increase of nitric oxide (NO), forthe treatment of hypertension or atherosclerosis. In addition, theinventors specifically envision the topical application of thetriterpene compounds of the invention for enhanced penile function. Alsocontemplated by the inventors is the topical administration of thecompounds of the invention for increasing skin collagen, therebycombating the effects of skin aging.

IX. Assays and Methods for Screening Active Compounds

A number of assays are known to those of skill in the art and may beused to further characterize the triterpene compounds of the invention.These include assays of biological activities as well as assays ofchemical properties. The results of these assays provide importantinferences as to the properties of compounds as well as their potentialapplications in treating human or other mammalian patients. Assaysdeemed to be of particular utility in this regard include in vivo and invitro screens of biological activity and immunoassays.

In Vivo Assays

The present invention encompasses the use of various animal models.Here, the identity seen between human and mouse provides an excellentopportunity to examine the function of a potential therapeutic agent,for example, a triterpene compound of the current invention. One canutilize cancer models in mice that will be highly predictive of cancersin humans and other mammals. These models may employ the orthotopic orsystemic administration of tumor cells to mimic primary and/ormetastatic cancers. Alternatively, one may induce cancers in animals byproviding agents known to be responsible for certain events associatedwith malignant transformation and/or tumor progression.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route the could be utilized for clinical or non-clinicalpurposes, including but not limited to oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumoral injection.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, survival, reduction of tumor burden or mass, arrest orslowing of tumor progression, elimination of tumors, inhibition orprevention of metastasis, increased activity level, improvement inimmune effector function and improved food intake.

One particularly useful type of in vivo assay of anti-tumor activitycomprises the use of a mouse skin model. The mouse skin model, whichrepresents one of the best understood experimental models of multistagecarcinogenesis, has permitted the resolution of three distinct stages inthe development of cancer: initiation, promotion, and progression. It isnow apparent that the cellular evolution to malignancy involves thesequential alteration of proto-oncogenes and/or tumor suppressor genes,whose gene products participate in critical pathways for thetransduction of signals and/or regulation of gene expression. The skintumor promotion and progression stages are characterized by selectiveand sustained hyperplasia, differentiation alterations, and geneticinstability leading to specific expansion of the initiated cells intopapillomas and carcinomas. It has been indicated that the induction of asustained hyperplasia correlates well with the skin tumor promotingactivity of various agents such as phorbol esters, several peroxides,and chrysarobin. In the mouse skin model all known carcinogens and tumorpromoters have been shown to produce a sustained epidermal hyperplasia.In general, this is preceded by an inflammatory response.

Extensive data has revealed a good correlation between carcinogenicityand mutagenicity. Most tumor-initiating agents either generate or aremetabolically converted to electrophilic reactants, which bindcovalently to cellular DNA. Some free radicals and modified DNA basesare free radicals have been implicated in the tumor initiation and/ortumor promotion stages of carcinogenesis. Strong evidence has indicatedthat activation of the Ha-ras gene occurs early in the process of mouseskin carcinogenesis and perhaps is equivalent to an initiation event.For example, it has been shown that the presence of an activatedc-Ha-ras gene in mouse skin papillomas and carcinomas induced by7,12-dimethylbenz[a]anthracene was associated with a high frequency ofA-T transversions at codon 61. Subsequent studies demonstrated that thistype of mutation was dependent upon the chemical initiator andindependent of the promoter, suggesting a direct effect of the initiatoron c-Ha-ras. Furthermore, infection of mouse skin by a virally activatedHa-ras gene (v-Ha-ras) can serve as the initiating even in two-stagecarcinogenesis. It should be emphasized that all skin chemicalcarcinogens and skin tumor initiators have been shown to produce amutation in Ha-ras oncogene. However, skin tumor promoters do not causea mutation in Ha-ras.

(ii) Confirmatory In Vivo and Clinical Studies

It will be understood by those of skill in the art that chemotherapeuticagents, including the triterpene compounds of the invention, orcombinations of such with additional agents, should generally be testedin an in vivo setting prior to use in a human subject. Such pre-clinicaltesting in animals is routine in the art. To conduct such confirmatorytests, all that is required is an art-accepted animal model of thedisease in question, such as an animal bearing a solid tumor. Any animalmay be used in such a context, such as, e.g., a mouse, rat, guinea pig,hamster, rabbit, dog, chimpanzee, or such like. In the context of cancertreatment, studies using small animals such as mice are widely acceptedas being predictive of clinical efficacy in humans, and such animalmodels are therefore preferred in the context of the present inventionas they are readily available and relatively inexpensive, at least incomparison to other experimental animals.

The manner of conducting an experimental animal test will bestraightforward to those of ordinary skill in the art. All that isrequired to conduct such a test is to establish equivalent treatmentgroups, and to administer the test compounds to one group while variouscontrol studies are conducted in parallel on the equivalent animals inthe remaining group or groups. One monitors the animals during thecourse of the study and, ultimately, one sacrifices the animals toanalyze the effects of the treatment.

One of the most useful features of the present invention is itsapplication to the treatment of cancer. Accordingly, anti-tumor studiescan be conducted to determine the specific effects upon the tumorvasculature and the anti-tumor effects overall. As part of such studies,the specificity of the effects should also be monitored, including thegeneral well being of the animals.

In the context of the treatment of solid tumors, it is contemplated thateffective amounts of the triterpene compounds of the invention will bethose that generally result in at least about 10% of the cells within atumor exhibiting cell death or apoptosis. Preferably, at least about20%, about 30%, about 40%, or about 50%, of the cells at a particulartumor site will be killed. Most preferably, 100% of the cells at atuinor site will be killed.

The extent of cell death in a tumor is assessed relative to themaintenance of healthy tissues in all of the areas of the body. It willbe preferable to use doses of the compounds of the invention capable ofinducing at least about 60%, about 70%, about 80%, about 85%, about 90%,about 95% up to and including 100% tumor necrosis, so long as the dosesused do not result in significant side effects or other untowardreactions in the animal. All such determinations can be readily made andproperly assessed by those of ordinary skill in the art. For example,attendants, scientists and physicians can utilize such data fromexperimental animals in the optimization of appropriate doses for humantreatment. In subjects with advanced disease, a certain degree of sideeffects can be tolerated. However, patients in the early stages ofdisease can be treated with more moderate doses in order to obtain asignificant therapeutic effect in the absence of side effects. Theeffects observed in such experimental animal studies should preferablybe statistically significant over the control levels and should bereproducible from study to study.

Those of ordinary skill in the art will further understand thatcombinations and doses of the compounds of the invention that result intumor-specific necrosis towards the lower end of the effective rangesmay nonetheless still be useful in connection with the presentinvention. For example, in embodiments where a continued application ofthe active agents is contemplated, an initial dose that results in onlyabout 10% necrosis will nonetheless be useful, particularly as it isoften observed that this initial reduction “primes” the tumor to furtherdestructive assault upon subsequent re-application of the therapy. Inany event, even if upwards of about 40% or so tumor inhibition is notultimately achieved, it will be understood that any induction ofthrombosis and necrosis is nonetheless useful in that it represents anadvance over the state of the patients prior to treatments. Stillfurther, it is contemplated that a dose of the compounds of theinvention which prevents or decreases the likelihood of eithermetastasis or de novo carcinogenesis would also be of therapeuticbenefit to a patient receiving the treatment.

As discussed above in connection with the in vitro test system, it willnaturally be understood that combinations of agents intended for usetogether should be tested and optimized together. The compounds of theinvention can be straightforwardly analyzed in combination with one ormore chemotherapeutic drugs, immunotoxins, coaguligands or such like.Analysis of the combined effects of such agents would be determined andassessed according to the guidelines set forth above.

(iii) In Vitro Assays

In one embodiment of the invention, screening of plant extracts isconducted in vitro to identify those compounds capable of inhibiting thegrowth of or killing tumor cells. Killing of tumor cells, orcytotoxicity, is generally exhibited by necrosis or apoptosis. Necrosisis a relatively common pathway triggered by external signals. Duringthis process, the integrity of the cellular membrane and cellularcompartments is lost. On the other hand, apoptosis, or programmed celldeath, is a highly organized process of morphological events that issynchronized by the activation and deactivation of specific genes(Thompson et al., 1992; Wyllie, 1985).

An efficacious means for in vitro assaying of cytoxicity comprises thesystematic exposure of a panel of tumor cells to selected plantextracts. Such assays and tumor cell lines suitable for implementing theassays are well known to those of skill in the art. Particularlybeneficial human tumor cell lines for use in in vitro assays ofanti-tumor activity include the human ovarian cancer cell lines SKOV-3,HEY, OCC 1, and OVCAR-3; Jurkat T-leukemic cells; the MDA-468 humanbreast cancer line; LNCaP human prostate cancer cells, human melanomatumor lines A375-M and Hs294t; and human renal cancer cells 769-P,786-0, A498. A preferred type of normal cell line for use as a controlconstitutes human FS or Hs27 foreskin fibroblast cells.

In vitro determinations of the efficacy of a compound in killing tumorcells may be achieved, for example, by assays of the expression andinduction of various genes involved in cell-cycle arrest (p21, p27;inhibitors of cyclin dependent kinases) and apoptosis (bcl-2, bcl-x_(L)and bax). To carry out this assay, cells are treated with the testcompound, lysed, the proteins isolated, and then resolved on SDS-PAGEgels and the gel-bound proteins transferred to nitrocellulose membranes.The membranes are first probed with the primary antibodies (e.g.,antibodies to p21, p27, bax, bcl-2 and bcl-x_(l), etc.) and thendetected with diluted horseradish peroxidase conjugated secondaryantibodies, and the membrane exposed to ECL detection reagent followedby visualization on ECL-photographic film. Through analysis of therelative proportion of the proteins, estimates may be made regarding thepercent of cells in a given stage, for example, the G0/G1 phase, S phaseor G2/M phase.

Cytotoxicity of a compound to cancer cells also can be efficientlydiscerned in vitro using MTT or crystal violet staining. In this method,cells are plated, exposed to varying concentrations of the samplecompounds, incubated, and stained with either MTT(3-(4,5-dimethylethiazol-2-yl)-2,5-diphenyle tetrazolium bromide; SigmaChemical Co.) or crystal violet. MTT treated plates receive lysis buffer(20% sodium dodecyl sulfate in 50%

DMF) and are subject to an additional incubation before taking an ODreading at 570 nm. Crystal violet plates are washed to extract dye withSorenson's buffer (0.1 M sodium citrate (pH 4.2), 50% v/v ethanol), andread at 570-600 nm (Mujoo et al., 1996). The relative absorbanceprovides a measure of the resultant cytotoxicity.

(iv) Immunoassays

Immunoassays may find use with the current invention, for example, inthe screening of extracts from plant species other than Acacia victoriaefor the triterpene compounds of the invention. Immunoassays encompassedby the present invention include, but are not limited to those describedin U.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich assay)and U.S. Pat. No. 4,452,901 (Western blot). Other assays includeimmunoprecipitation of labeled ligands and immunocytochemistry, both invitro and in vivo.

Immunoassays, in their most simple and direct sense, are binding assays.Certain preferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in theart. Immunohistochemical detection using tissue sections also isparticularly useful.

In one exemplary ELISA, anti-triterpene antibodies are immobilized ontoa selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining the triterpene compounds of the instant invention, such as aplant extract from a plant related to Acacia victoriae, is added to thewells. After binding and washing to remove non-specifically bound immunecomplexes, the bound antigen may be detected. Detection is generallyachieved by the addition of another antibody specific for the desiredantigen and which is linked to a detectable label. This type of ELISA isa simple “sandwich ELISA”. Detection also may be achieved by theaddition of a second antibody specific for the desired antigen, followedby the addition of a third antibody that has binding affinity for thesecond antibody, with the third antibody being linked to a detectablelabel.

Variations of ELISA techniques are know to those of skill in the art. Inone such variation, the samples suspected of containing the desiredantigen are immobilized onto the well surface and then contacted withthe prepared antibodies. After binding and appropriate washing, thebound immune complexes are detected. Where the initial antigen specificantibodies are linked to a detectable label, the immune complexes may bedetected directly. Again, the immune complexes may be detected using asecond antibody that has binding affinity for the first antigen specificantibody, with the second antibody being linked to a detectable label.

Competition ELISAs also are possible in which test samples compete forbinding with known amounts of labeled antigens or antibodies. The amountof reactive species in the unknown sample is determined by mixing thesample with the known labeled species before or during incubation withcoated wells. The presence of reactive species in the sample acts toreduce the amount of labeled species available for binding to the welland thus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described as below.

Antigen or antibodies also may be linked to a solid support, such as inthe form of plate, beads, dipstick, membrane or column matrix, and thesample to be analyzed applied to the immobilized antigen or antibody. Incoating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period. The wells of theplate will then be washed to remove incompletely adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein andsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding ofthe antigen or antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the clinical or biologicalsample to be tested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, or a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG) and phosphate buffered saline (PBS)/Tween. These addedagents also tend to assist in the reduction of nonspecific background.

The suitable conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours, attemperatures preferably on the order of 25° to 27° C., or may beovernight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. Washing often includeswashing with a solution of PBS/Tween, or borate buffer. Following theformation of specific immune complexes between the test sample and theoriginally bound material, and subsequent washing, the occurrence ofeven minute amounts of immune complexes may be determined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact andincubate the first or second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation, e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween.

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2’-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer. Alternatively, the label maybe a chemiluminescent one. The use of such labels is described in U.S.Pat. Nos. 5,310,687, 5,238,808 and 5,221,605.

Methods for in vitro and in situ analysis are well known and involveassessing binding of antigen-specific antibodies to tissues, cells orcell extracts. These are conventional techniques well within the graspof those skilled in the art. For example, the antibodies to tumor cellantigens may be used in conjunction with both fresh-frozen andformalin-fixed, paraffin-embedded tissue blocks prepared for study byimmunohistochemistry (IHC). Each tissue block may consist of 50 mg ofresidual “pulverized” tumor. The method of preparing tissue blocks fromthese particulate specimens has been successfully used in previous IHCstudies of various prognostic factors, e.g., in breast cancer, and iswell known to those of skill in the art.

Briefly, frozen-sections may be prepared by rehydrating 50 ng of frozenpulverized tumor at room temperature in PBS in small plastic capsules;pelleting the particles by centrifugation; resuspending them in aviscous embedding medium (OCT); inverting the capsule and pelletingagain by centrifugation; snap-freezing in −70° C. isopentane; cuttingthe plastic capsule and removing the frozen cylinder of tissue; securingthe tissue cylinder on a cryostat microtome chuck; and cutting 25-50serial sections containing an average of about 500 remarkably intacttumor cells.

Permanent-sections may be prepared by a similar method involvingrehydration of the 50 mg sample in a plastic microfuge tube; pelleting;resuspending in 10% formalin for 4 hours fixation; washing/pelleting;resuspending in warm 2.5% agar; pelleting; cooling in ice water toharden the agar; removing the tissue/agar block from the tube;infiltrating and embedding the block in paraffin; and cutting up to 50serial permanent sections.

In light of the present disclosure, one could utilize screening assaysfor the identification of compounds having essentially the same chemicalcharacteristics and biological activity as those described herein. Inparticular, the present disclosure would allow one to employ assays forbiologically active triterpene glycosides from those plants closelyrelated to Acacia victoriae, for example, members of the genus Acacia.These assays may make use of a variety of different formats' and maydepend on the kind of “activity” for which the screen is beingconducted. Preferred assays comprise those directed to screening foranti-tumor activity, such as described herein for extracts from Acaciavictoriae. As used herein, “anti-tumor activity” refers to theinhibition in tumor cells of cell-to-cell signaling, growth, metastasis,cell division, cell migration, soft agar colony formation, contactinhibition, invasiveness, angiogenesis, tumor progression or othermalignant phenotype or the induction of apoptosis. Particularlycontemplated are functional assays which include measures of the use ofthe compounds of the invention as anti-fungal and anti-viral agents,piscicides or molluscicides, contaceptives, anthelmintics,UV-protectants, expectorants, diuretics, anti-inflammatory agents,regulators of cholesterol metabolism, cardiovascular effectors,anti-ulcer agents, analgesics, sedatives, immunomodulators,antipyretics, regulators of angiogenesis, and as agents for decreasingcapillary fragility. Such assays will be well known to those of skill inthe art in light of the instant disclosure. As well as in vitro and invivo direct assays for activity, these assays may include measures ofinhibition of binding to a substrate, ligand, receptor or other bindingpartner by a compound of the invention.

X. Growth and Tissue Cultures of Acacia victoriae

An important aspect in the preparation of the compounds of the inventionwill be the availability of tissue of Acacia victoriae. As the inventorshave shown that the compounds of the invention are concentrated in rootsand pods of Acacia victoriae, the availability of these tissues will beparticularly important. The inventors have also shown that youngseedlings, are another source for isolating the compounds of thisinvention. Acacia victoriae grows in the southwest United States and inAustralia, and therefore, plant tissue is available to the public.Additionally, a deposit of 2500 seeds of Acacia victoriae has been madeby the inventors with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 on (May 7, 1998). Thosedeposited seeds have been assigned ATCC Accession No. 209835. Thedeposit was made in accordance with the terms and provisions of theBudapest Treaty relating to deposit of microorganisms and is made for aterm of at least thirty (30) years and at least five (05) years afterthe most recent request for the furnishing of a sample of the depositwas received by the depository, or for the effective term of the patent,whichever is longer, and will be replaced if it becomes non-viableduring that period.

Therefore, in light of the instant disclosure, one of skill in the artcould plant those deposited seeds, grow plants therefrom, and isolatetissue from the plants for the preparation of the triterpene compoundsand nutraceuticals of the invention. Also, one could isolate tissue fromnaturally occurring Acacia victoriae populations. However, thepreparation of tissue for isolation of the compounds of the inventionwould be more readily achieved if a suitable cultivation technique weredesigned for the propagation of Acacia victoriae tissue. One option forthe preparation of tissue would be the large-scale cultivation of thespecies. More preferable options, however, include tissue cultures ofAcacia victoriae and implementation of an aeroponic growth system.

Aeroponic Growth Techniques

A number of advantages may be realized by utilization of an aeroponicsystem of cultivation for Acacia victoriae. First; the growth rate ofthe plants is approximately twice that achieved with conventionalgrowing techniques. Second, the roots can be easily harvested as neededwithout harming the plants. The cutting of roots further leads toextensive lateral growth of fibrous roots. Therefore, the roots could beharvested several time a year. In wild populations of Acacia victoriae,collection of pods is limited to several weeks a year, and collection ofroots is difficult without harming or killing the plant.

An aeroponic growth system is a closed system in which plant roots aresuspended in air and misted with a complete nutrient solution. The rootsare enclosed in a watertight box misted at intervals with the nutrientsolution. The nutrient solution contains all of the essential elementsthe plants needs to complete its life cycle. Despite the fact thatdifferent plants require different levels and formulations for optimumgrowth, an over-all, single-balanced solution gives satisfactoryresults.

(ii) Tissue Cultures of Acacia victoriae

Tissue cultures represent another option for cultivation of Acaciavictoriae. For the development of tissue cultures, Acacia victoriaeseeds are washed thoroughly in tap water with an anti-microbial soap andtreated with a 20% solution of commercial bleach for 15 minutes. Afterrepeated washing in deionized water, the seeds are treated with boilingwater to induce germination and incubated overnight. The next morning,seeds are once again disinfected with commercial bleach and rinsed 2-3times in sterile deionized water. The decontaminated seeds are thencultured on MS medium (Murashige et al., 1962) supplemented with MSvitamins and 2% sucrose (for the explant cultures, 3% sucrose was used)and the medium gelled with either 0.7% agar or 0.2% gelrite.

Explants used for culturing may comprise potentially any tissue typeincluding shoot tips, nodal segments, hypocotyls and root segments. Theexplants are generally cultured on MS alone or MS supplemented withgrowth regulators, such as IAA, NAA, IBA, 2,4-D and BAP (eitherindividually or in combination). The cultures are typically maintainedat 25±2° C. under a 16 hour light photoperiod at 1000 lux produced bycool white fluorescent tubes. Resulting plantlets are kept under mist inthe green house one month for hardening before transferring them to agreenhouse, field or aeroponic growth system.

Hairy root cultures of Acacia victoriae have been developed in thepresent invention. Infection of the plant with Agrobacterium rhizogenesstrain R-1000, leads to the integration and expression of T-DNA in theplant genome, which causes development of a hairy roots. Hairy rootcultures grow rapidly, show plagiotropic root growth and are highlybranched on hormone-free medium and also exhibit a high degree ofgenetic stability (Aird et al., 1988). The genetic transformation andinduction of hairy roots in Acacia victoriae and the optimum conditionsfor growth are described in detail in the section on Examples. Hairyroot cultures allow the rapid growth of tissue on a large scale whichcan be used for the isolation of the triterpene compounds of thisinvention.

An advantage of tissue culturing is that clonal cultures may potentiallybe prepared which express the compounds of the invention. These culturescould be grown on a large scale and potentially be expanded to anindustrial capacity growth system for the preparation of plant tissuefor the isolation of triterpene compounds. Additionally, plantsregenerated from tissue cultures frequently display significantvariation. Therefore, using tissue cultures, clonal cell lines or plantsregenerated from such cultures may be produced which are “elite” withregard to their production of the triterpene compounds of the invention.Plants produced could be selfed over generation and selected at eachbreeding generation to produce true-breeding elite lines.

Elite varieties need not necessarily arise from tissue cultures,however, as significant genetic variation exists within wild populationsof Acacia victoriae. It is, therefore, contemplated by the inventorsthat the genetic variation found in wild populations of Acacia victoriaeincludes variations in genes controlling the endogenous levels oftriterpene production. As such, it should be possible to identify thosemembers of Acacia victoriae populations which produce enhanced levels oftriterpenes relative to other members of wild populations, and to selectthese varieties for use in growth systems directed to producing tissuefor the isolation of the triterpene compounds of the invention. Thegrowth system may constitute, for example, convention farming, aeroponicgrowth techniques, tissue culturing, or any other suitable technique forthe propagation of Acacia victoriae tissue. Still further, these plantsmay be selected for use in breeding protocols to produce varieties whichare more elite and which are also true-breeding.

XI. Definitions

“A” means “one or more.” Thus, a moiety may refer to one, two, three, ormore moieties.

Active constituents refers to the most pure extract that retainsactivity. In the present invention, the “active component” or “activecompound” refers to the active triterpene compounds identified by theinstant inventors. These compounds have been purified and identified in,for example, fraction UA-BRF-004-DELEP-F094.

Pods are defined as seedpods of Acacia victoriae.

Cytotoxic is defined as cell death while the term “cytostatic” isdefined as an inhibition of growth and/or proliferation of cells.

Apoptosis is defined as a normal physiologic process of programmed celldeath which occurs during embryonic development and during maintenanceof tissue homeostasis. The process of apoptosis can be subdivided into aseries of metabolic changes in apoptotic cells. Individual enzymaticsteps of several regulatory or signal transduction pathways can beassayed to demonstrate that apoptosis is occurring in a cell or cellpopulation, or that the process of cell death is disrupted in cancercells. The apoptotic program is also observed by morphological featureswhich include changes in the plasma membrane (such as loss ofasymmetry), a condensation of the cytoplasm and nucleus, andinternucleosomal cleavage of DNA. This is culminated in cell death asthe cell degenerates into “apoptotic bodies”.

Techniques to assay several enzymatic and signaling processes involvedin apoptosis have been developed as standard protocols formultiparameter apoptosis research. One example of an early step inapoptosis, is the release of cytochrome c from mitochondria followed bythe activation of the caspase-3 pathway (PharMingen, San Diego, Calif.).Induction of the caspases (a series of cytosolic proteases) is one ofthe most consistently observed features of apoptosis. In particular,caspase-3 plays a central role in the process. When caspases areactivated, they cleave target proteins; one of the most important ofthese is PARP (poly-(ADP-ribose) polymerase, which is a protein locatedin the nucleus). Therefore, assays detecting release of cytochrome c,detecting caspase-3 activity and detecting PARP degradation areeffective determinants of apoptosis.

Furthermore, agents that cause the release of cytochrome c from themitochondria of malignant cells can be concluded to be likely therapiesfor restoring at least some aspects of cellular control of programmedcell death.

Another apoptotic assay is the Annexin-V detection (BioWhitaker,Walkerville, Md.). Normally, phosphotidylserine (PS) is localized on theinner membrane of the plasma membrane. However, during the early stagesof apoptosis, externalization of PS takes place. Annexin-V is a calciumbinding protein which binds to PS and can be observed withannexin-V-FITC staining by flow cytometry (Martin et al., 1995). Theability of cells treated with the Acacia victoriae compounds describedin this invention, to bind annexin-V, is taken as an indication thatcells were undergoing apoptosis.

In other examples, the inventors have used PI-3-Kinase assay and todetect the apoptotic activity in cells treated with mixtures of theanti-cancer compounds isolated from Acacia victoriae. Phosphoinositide3-kinase (PI3K), a cell membrane associated enzyme, is capable ofphosphorylating the 3-position of the inositol ring ofphosphatidylinositol, thus defining a new lipid signaling pathway inthose cells where PI3K is active. When PI3K is active, a kinase calledAKT is recruited to the cell membrane. AKT is the product of an oncogenewhich is catalytically activated after recruitment to the membrane.Fully activated AKT plays a crucial role in cell survival. The PI3K/AKTpathway provides a mechanism by which cells evade apoptosis. Thus, ameans to inhibit PI3K in malignant cells, is a likely therapy forrestoring at least some aspects of the cellular control of apoptosis.

Abnormal Proliferation is defined as a series of genetically determinedchanges that occur in mammalian cells in the pathological state known ascancer. This process eventually results in the loss of control ofapoptosis in cancer cells. This can occur in steps, generally referredto as 1. initiation, which is defined as the stage when an externalagent or stimulus triggers a genetic change in one or more cells, and 2.promotion, which is defined as the stage involving further genetic andmetabolic changes, which can include inflamation. During the “promotionstage”, cells begin a metabolic transition to a stage of cellular growthin which apoptosis is blocked.

Malignant cells are defined as cancer cells that escape normal growthcontrol mechanisms through a series of metabolic changes during theinitiation and promotion stages of the onset of malignancy. Thesechanges are a consequence of genetic alterations in the cells (eitheractivating mutations and/or increased expression ofprotooncogenes—and/or inactivating mutations and/or decreased expressionof one or more tumor suppressor genes). Most oncogene and tumorsuppressor gene products are components of signal transduction pathwaysthat control cell cycle entry or exit, promote differentiation, senseDNA damage and initiate repair mechanisms, and/or regulate cell deathprograms. Cells employ multiple parallel mechanisms to regulate cellgrowth, differentiation, DNA damage control, and apoptosis. Nearly alltumor and malignant cells have mutations in multiple oncogenes and tumorsuppressor genes.

Extract or fraction refers to consecutive samples collected from tissuesby various means. These “extracts” or “fractions” may be analyzed forthe desired anti-tumor activity, and further “extracted” or“fractionated” to produce successively more pure componentscorresponding to the, active component.

Triterpene or Triterpene Glycoside refers to the novel and/orbiologically active saponin compounds identified herein from Acaciavictoriae. The triterpene or triterpene glycosides need not be isolatedfrom Acacia victoriae, as one of skill in the art, in light of theinstant disclosure, could isolate the compounds from related species, orchemically synthesize analogs of the triterpenes and triterpeneglycosides disclosed herein. “Triterpenes” of this invention include thesaponin compounds described herein which have at least a triterpeneunit(s) and, in the case of triterpene glycosides, a sugar orsaccharide. These terms also refer to compounds containing additionalmoieties or chemical functionalities including, but not limited to,monoterpene units as will be apparent from the rest of thespecification. Thus, triterpenes of this invention also include theaglycones formed by hydrolysis of sugar units and further includes othermodification of the triterpenoid compounds, whereby the modifications donot destroy the biological activity of the compounds.

XII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Preliminary Screening and Purification of Anti-Tumor ActiveConstituents from Acacia victoriae

Sixty plant species were chosen from the Desert Legume Project (DELEP)with the goal of identifying novel compounds having beneficialbiological activities. The DELEP (University of Arizona, Tucson) is acollection of desert legume species developed through a collaborationbetween the University of Arizona and the Boyce Thompson SouthwesternArboretum. Experimental field samples were collected from each of theplant species, air-dried for 3-4 days, ground to three millimeterparticle size with a Wiley mill (3 mm screen size) and extracted two orthree times by percolation with a 1:1 mixture of dichloromethane (DCM)and methanol (MeOH). Each percolation extraction proceeded for at least5 hours and often continued overnight. The majority of the extractedbiomass was collected from the first two percolations. The biomass wasthen washed with a volume of methanol equal to half the void volume, andthe crude extract contained in the methanol aliquots isolated. Thesamples were typically isolated and prepared for bioassay by removingthe methanol in vacuo, passing the aqueous phase through RP-C 18particles, recovering the active constituents in MeOH, and thenrotovapping the MeOH to collect the extract as a solid. The crudeextract was then resuspended in H20, DMSO or mixtures thereof (lesspolar compounds were resuspended in DMSO, while more polar compoundswere resuspended in water or water and DMSO mixtures; aglycones wereresuspended in DMSO).

Each of the extracts was then screened against a panel of human tumorand non-tumor cells including human ovarian cancer cell lines,T-leukemic cells, human epidermoid cells, human breast cancer cells,human prostate cancer cells, human foreskin fibroblast cells, humanendothelial cells, and human renal cancer cells. The cells were firstplated in 96-well plates for 18-24 hours at 37° C. The_cells were thenexposed to varying concentrations of the plant extracts, and incubatedfor 72 hours at 37° C., and stained with either MTT(3-(4,5-dimethylethiazol-2-yl)-2,5-diphenyl tetrazolium bromide; SigmaChemical Co.) for 4 hours or crystal violet (Sigma Chemical Co.) for 20minutes at room temperature. The MIT plates received lysis buffer (20%sodium dodecyl sulfate in 50% DMF) and were incubated for an additional6 hours before taking an OD reading at 570 nm. The crystal violet plateswere washed, dye was extracted for 3-4 hours with Sorenson's buffer (0.1M sodium citrate (pH 4.2), 50% v/v ethanol), and the plates were read at570-600 nm (Mujoo et al., 1996). Cytotoxicity of the screened extractswas indicated by comparing the OD readings between the treated mediaalone and the cells treated with the plant extract. Percent cytotoxicitywas calculated by 100-% of control, where % of control=[((OD of cellstreated with plant extract (treated sample))/(OD of cells exposed tomedia alone (untreated sample)))×100].

Of the initial screening, one plant extract showed potent growthinhibition of cancer cells while demonstrating little toxicity to normalhuman fibroblast cells. This extract, coded UA-BRF-004-DELEP-F001, wasisolated from the leguminous plant Acacia victoriae. The extractexhibited an IC₅₀ at approximately 12 μg/ml (SKOV-3) 26 μg/ml (OVCAR-3)and 13 μg/ml (HEY) using human ovarian cancer cell lines; at greaterthan 50 μg/ml (A375-M) and at about 38 μg/ml (HS294T) with humanmelanoma cells; at about 15 μg/ml for human epidermoid cells (A431); andat greater than 50 μg/ml for the breast cancer cell line MDA-468(FIG. 1) (see Example 13 for a description of cell lines). Among normalhuman foreskin fibroblast cells (FS) and mouse fibroblast cells (L929)treated with the same extract, no cytoxicity was observed.

This extract appeared to contain a mixture of many constituents by TLC.Therefore, preliminary efforts focused on purifying this extract toisolate the active constituents responsible for the selectivecytotoxicity. Chromatographic fractions enriched in the activeconstituents were isolated from original extract according to thegeneral scheme shown in FIG. 15.

The original extract, UA-BRF-004-DELEP-F001, was prepared from 538 g ofplant material from Acacia victoriae by percolation (twice) as describedabove. The extract was then dried in vacuo yielding approximately 52.0 gof powder. Then, 51.5 g of the dried material was treated 3 times with 1L ethyl acetate (“EtOAc”). Approximately 15.75 g of the EtOAc solublematerial was subject to column chromatography on silica gel (1.5 kg).Fifty-four 670 ml subfractions eluted employing increasingly polarmixtures of hexane, EtOAc, and MeOH. The 54 subfractions were collectedinto thirteen separate fractions, labeled as UA-BRF-004-DELEP-F006 toUA-BRF-004-DELEP-F018. These fractions were then screened for anti-tumoractivity using the procedure described above. None of the fractionsexamined demonstrated the potent anti-tumor activity observed inUA-BRF-004-DELEP-F001.

The EtOAc insoluble material (approximately 34.7 g) was also subject tochromatography on silica gel (1.7 kg). Fifty-one 670 ml subfractions andthree additional subfractions totaling 21 L were eluted employingincreasingly polar mixtures of DCM, MeOH and water. These subfractionswere collected into eight separate fractions labeledUA-BRF-004-DELEP-F019 to UA-BRF-004-DELEP-F026, according to Table 6.

TABLE 6 Elution of fractions UA-BRF-004-DELEP-F019 toUA-BRF-004-DELEP-F026 Total Fraction Collected From Weight IdentifierSubfractions¹ (mg) Eluent F019  1-13 1015 5% MeOH/DCM (1-6) 10% MeOH/DCM(7-12) 20% MeOH/DCM (13) F020 14-16 723 20% MeOH/DCM F021 17-19 3080 20%MeOH/DCM (17-18) 35% MeOH/DCM (19) F022 20-22 4618 35% MeOH/DCM F02323-34 17216 35%-50% MeOH/DCM (23-34) 39-40 65% MeOH/DCM (39) 100% MeOH(40) F024 35-38 3030 65% MeOH/DCM (35-38) 41-51 100% MeOH (41-51) F025 9L and 6 L 3980 MeOH (9 L) subfractions 20% water/MeOH (6 L) F026 6 Lsubfraction 4507 20% water and 1% HCOOH in MeOH ¹Each subfractionconsisted of 670 ml unless otherwise indicated.

Each of the fractions were then screened for anti-tumor activity againsta panel of human tumor cells as described above for the crude extract.One of the fractions, UA-BRF-004-DELEP-F023, exhibited an anti-tumoractivity which was more potent than that of UA-BRF-004-DELEP-F001. Theseresults revealed that 6 μg/ml of fraction UA-BRF-004-DELEP-F023exhibited 50% (OCCl), 63% (SKOV-3), 85% (HEY), and 48% (OVCAR-3)cytotoxicity on human ovarian cancer cells; approximately 60%cytotoxicity on human prostate cancer cells (LNCaP); about 92%cytoxicity on leukemic cells (Jurkat) and about 73% cytoxicity on freshhuman ovarian cancer cells from the ascites of patients (FTC). Bioassaysof non-transformed cells revealed an IC₅₀ of 10.6 μg/ml for FS cells and23 μg/ml for HUVEC cells (FIG. 2).

The biologically active component(s) in UA-BRF-004-DELEP-F023 werefurther purified by multiple reversed phase mode (RP) medium pressureliquid chromatographic (MPLC) separations to aid in the isolation andcharacterization of the active component(s). The samples, were elutedfrom degassed mixtures of increasing concentrations of acetonitrile(ACN) in water in 4 L increments of 10% according to the followingsteps: 0, 10%, 20%, 30%, 40% ACN/water. Then a 2-4 L fraction was elutedwith MeOH. Ten fractions were collected after repeated runs, labeledUA-BRF-004-DELEP-F027 to UA-BRF-004-DELEP-F036, according to Table 7.

TABLE 7 Elution of Fractions UA-BRF-004-DELEP-F027 toUA-BRF-004-DELEP-F036 Fraction Identifier Total Weight (g) Eluent F0276.95  0-20% ACN in water F028 0.99 30-40% ACN in water F029 1.46 30-40%ACN in water F030 0.86 30-40% ACN in water F031 0.15 30-40% ACN in waterF032 1.01 30-40% ACN in water F033 0.54 30-40% ACN in water F034 0.5030-40% ACN in water F035 2.19 30-40% ACN in water F036 1.17 30-40% ACNin water

Several of these fractions appeared similar by TLC. One'of the higheryielding fractions, UA-BRF-004-DELEP-F035 (Fraction 35), was found toexhibit potent anti-tumor activity.

The screening of UA-BRF-004-DELEP-F035 for anti-tumor activity revealedan IC₅₀ at 3.0, 1.2, 2.0 and 3.5 μg/ml, respectively, against theovarian cancer cell lines HEY, SKOV-3, OVCAR-3 and C-1 (cisplatinresistant OVCAR-3); an IC₅₀ of 2.4 μg/ml against pancreatic cancer cells(Panc-1); an IC₅₀ of 1.2 pg/ml, 3.0 μg/ml, and 3.7 μg/ml, respectivelyfor the renal cancer cell lines 769-P, 786-0, and A498; an IC₅₀ of 130ng/ml for Jurkat T-leukemic cells; and an IC₅₀ between 1-3 μg/ml for theB-leukemic cell lines KG1, REH and NALM-6 (FIG. 3, FIG. 4). As shown inTable 8, purification of the crude plant extract increased thebioactivity dramatically.

TABLE 8 Cytotoxicity Of Crude Extract Versus UA-BRF-004-DELEP-F035 IC₅₀(μg/ml) Human Cancer Cells crude extract UA-BRF-004-DELEP-F035 HEY 123.0 SKOV-3 25 1.2 OVCAR-3 25 2.0 MDA-468 50 9.0

Fraction 35 exhibited an IC₅₀ of approximately 4.7 μg/ml to normal humanFS cells and an IC₅₀ of approximately 13.3 μg/ml to normal human Hs27cells. When the effect of Fraction 35 (F035) was evaluated on normalhuman erythroid and myleoid colonies (cells isolated from bone marrow),12-18% inhibition was observed at 3.0 μg/ml (Table 9).

TABLE 9 Effect of Fraction 35 on Erythroid and Myeloid ColoniesErythocyte Myleoid (# Percent (# of Percent of colonies) inhibitioncolonies) Inhibition untreated 261 — 111 — F035 (30 μg/ml) 16 94 53 52F035 (3 μg/ml) 212 18 97 12 F035 (0.3 μg/ml) 248 5.0 119 7 (stimulation)In light of the above findings indicating the potent anti-tumor activityof Fraction 35, a bioassay was conducted as described above usingconcentrations of Fraction 35 as low as 0.095 μg/ml. In this study,varying concentrations of Fraction 35 were used against an expandedpanel of tumor lines. The results of the screening indicate that even atconcentrations of 1.56 μg/ml Fraction 35 exhibited potent anti-tumoractivity against a number of cell lines (Table 10).

TABLE 10 Cytotoxicity of Varying Concentrations ofUA-BRF-004-DELEP-9F035 Against Different Tumor Cell Lines. UA-BRF-004-25 12.5 0.095 DELEP-F035 50 μg/ml μg/ml μg/ml 6.25 μg/ml 3.12 μg/ml 1.56μg/ml 0.78 μg/ml 0.39 μg/ml 0.195 μg/ml μg/ml SKOV-3 94% 83.40% 78.50%  71%   54%   27%    0% 0% 0% OVCAR-3 95% 92.80%   91%   87%   79%   46%   9% 21.40%    18%  C-1 97%   71%   87%   77%   59%   29%    0% 0% 0%(OVCAR-3 VARIANT) HEY 97% 79.10% 53.90% 43.30% 19.20%    0%    0% 0% 0%C-2(HEY 96% 93.20% 90.50% 88.90% 86.80% 82.50% 73.40% 50%  36.10%   VARIANT) A-8(HEY 97.20%   95.70% 94.80%   89% 75.00% 59.30%   18% 0% 0%VARIANT) MCF-7 79.70%     23%    5%  3.10%  8.10% 17.20%  8.60%17.40%    19.20%    BT-20 83%   90%    0%    4% 12.50% 15.50% 21.30%24%  34%  MDA-MB-453 98.40%   97.20% 94.60% 89.90% 85.40% 81.60% 65.70%54.90%    38.50%    MDA-468 96.50%   93.80%   82%   65%   39%    8%   8% 8% 8% SKBR-3 83.90%   62.70% 51.70% 47.80% 45.20% 39.60% 35.90%28.60%    21.90%    PANC-1 97.30%   90.20% 66.80% 30.40%    0%    0%   0% 0% 0% 769-P 96.80%     97%   90%   95% 94.00% 91.70%   63% 18% 10%  17.80%    786-O 97.90%     89% 80.30%   75%   66%   32%    0% 0% 0%A-498 99%   97% 95.50%   80%   47%   19%   16% 15%  14%  LLC-1 84.70%  42.70% 17.80%  6.60% 10.70% 10.90%  3.80% 6.80%   0% A549 96.60%  91.10% 59.70% 34.80% 21.20% 15.60%    0% 0% 0% JURKAT 88.40%   88.70%88.80% 89.60% 88.60% 88.50%   80% 69.30%    46.60%    0% Hs27 88%   83%  47%    0%    6%   11%   11% 13%  18%  FS FIBRO 78.30%   74.70% 73.70%66.50% 42.30%    0%    0% 16%  23.20%    HL-60 63.00%   22.00% 30.00%25.00%  0.00%  0.00%  0.00% 0.00%   0% 0% MDA-MB-435 96.50%   96.40%  97%   96%   94% 84.70% 40.60% 15%  14.70%    DOV-13 95.80%   92.30%86.70% 77.80% 57.50% 14.20% 17.50% 11.10%    12.20%    17.20%    MCF-10A97.50%   11.70%    0%    1%    0%    0%    0% 0% 0% MCF-10F 97.70%   5.80%    0%    0%    0%    0%    0% 0% 0% KG-1 77%   75%   72%   67%  59%   44%   35% 13%  0% 0% OCl-2 46.40%     21%   12% 12.30%    9%  12%    3% 0% 0% 0% OCl-3 71%   60%   45%   41%   30%  5.60%    0% 0%0% 0%

Example 2 Procedures for Isolating Active Constituents from Acaciavictoriae

A procedure was developed for the direct preparation of fractionscontaining the active constituents contained in UA-BRF-004-DELEP-F035,isolated during the preliminary purification detailed in Example 1.Approximately 9665 g of freshly collected pod tissue from Acaciavictoriae was ground in a hammer mill with a 3 mm screen and thenextracted with 80% MeOH in H₂0 (3×) followed by filtration. 8200 g ofbagasse was discarded. The three washings were collected separately andassigned fraction identifiers as follows: F068 in 21.5 L (first wash);F069 in 24 L (second wash); and F070 in 34.3 L (third wash). F068 wasfurther purified by partitioning into 1 L aliquots, adding 400 ml H₂0 toeach aliquot and washing with CHCl₃ (2×250 ml). The combined polarphases (28.5 L) were assigned the fraction identifier of F078 and thecombined organic phases F079 (yielding 42.g after removal of the organicsolvent by rotovap).

The MeOH was removed from F078 in vacuo and F078 was furtherfractionated by RP MPLC using a 29×460 mm column loaded with 530 grecovered Bakerbond RP-C18, 40 μm particles. 500 ml of aqueous solutionwas aspirated onto the column and fractions collected according to Table11.

TABLE 11 Elution of Fractions F091 to F094. Fraction Volume TotalIdentifier Collected (L) Weight (g) Eluent Comments — 4 — 100% waterSugars and some strong RBC lysis component F091 4 ~40 10% ACN in 19.6 gobtained water from runs 16-29 F092 4 89 20% ACN in Flavonoids waterF093 4 351 30% ACN in Light fluffy solid, water slight respiratoryirritant F094 1.3 577 100% MeOH Fine powder, respiratory irritant

Each fraction was then desiccated by removal of MeOH, passing over C-18particles, recovering in MeOH, and isolated as a solid in vacuo. Thesolid was resuspended in water and subject to testing for anti-tumoreffects (for some less polar fractions, DMSO was added to the water;aglycones were resuspended in DMSO). The results indicated that thebiological activity of the 100% MeOH eluent, designated F094 wasessentially equivalent to that of fraction similarity of fractions F094and F035 was confirmed by TLC and HPLC, although F094 appeared tocontain additional components.

TABLE 12 Cytotoxicity of Varying Concentrations of F094 AgainstDifferent Tumor Cell Lines. UA-BRF-004Pod-DELEP- F094 50 μg/ml 25 μg/ml12.5 μg/ml 6.25 μg/ml 3.12 μg/ml 1.56 μg/ml 0.78 μg/ml 769-P 96.60%93.30% 92.80% 92.40% 88.30% 63.20% 21.80% PANC-1   97% 93.50% 74.60%50.60% 21.90%  1.10%    0% HEY   95% 66.50% 50.10% 17.90%    0%    0%   0% MDA-MB-453 94.20% 92.80% 87.10%   85% 77.30% 58.50%   47% JURKAT89.60% 89.80% 89.40% 89.30%   89%   88% 73.80%

F094 was further fractionated according to Table 13 and analyzed by TLCand bioassayed in order to obtain the purified active component(s). Theresults of the bioassay of varying amounts of the obtained fractions(F138-F147) are given in Table 14.

TABLE 13 Elution of Fractions F138 to F147. Fraction Subfractions TotalIdentifier Collected (ml)) Weight (g) Eluent —  1-5 (160) 1  60% MeOH inwater F138  6 (65) 13  60% MeOH in water (6) 7-8 (50)   70% MeOH inwater (7-8) F139  9 (25) 39  70% MeOH in water F140 10 (20) 93  70% MeOHin water F141 11 (35) 57  70% MeOH in water F142 12 (50) 54  70% MeOH inwater F143 13 (55) 62  70% MeOH in water F144 14 (70) 29  70% MeOH inwater F145 15 (65) 17  70% MeOH in water F146 16 (80) 54  80% MeOH inwater F147 17 (80) 7  80% MeOH in water (17)  18 (100) 100% MeOH inwater (18)

TABLE 14 Bioassay of Fractions F137, F140, F142, F144, and F145 50 μg/ml25 μg/ml 12.5 μg/ml F137 769-P 81.50 45.50 18.10 Panc-1 74 11 0 HEY 6.20 0 MDA-MB-453 76.70 38.80 26.80 JURKAT 67.70 67.50 67.80 F138 769-P96.50 95.60 95.30 Panc-1 95.50 93.45 73.50 HEY 65.30 58.30 21.50MDA-MB-453 96.10 94.20 92.5 JURKAT 87.50 88 87.50 F139 769-P 97.30 94.2094.20 Panc-1 96.60 94.10 86 HEY 89.70 65.80 60.50 MDA-MB-453 95 95 91.90JURKAT 88.50 88.50 88.50 F140 769-P 91.70 88.90 87.50 Panc-1 95 94.6092.50 HEY 95.40 72.10 62.80 MDA-MB-453 86.20 80.20 75.20 JURKAT 68.4067.80 68.10 F141 769-P 97.80 95.10 95 Panc-1 96.80 95 85.60 HEY 96 68.8060.6 MDA-MB-453 95 94.50 94 JURKAT 88.50 88.40 88 F142 769-P 92.50 90.2088.20 Panc-1 96 93.60 88.60 HEY 98 74.80 66 MDA-MB-453 86.10 75.40 72.90JURKAT 67.90 67.10 66.30 F143 769-P 98.30 96.80 98.30 Panc-1 96.70 94.7085.60 HEY 98.50 73 64 MDA-MB-453 96.70 95 94.10 JURKAT 88.00 88 88 F144769-P 89.80 88.60 89.50 Panc-1 96.60 93.80 90.90 HEY 98.50 75.30 62.20MDA-MB-453 86.70 78.50 75.80 JURKAT 65.70 65.70 65 F145 769-P 92 90.2086.30 Panc-1 96.70 91.40 84.80 HEY 97.50 82.30 58.60 MDA-MB-453 85.4074.40 48.90 JURKAT 67.90 68.40 68.60 F146 769-P 97.30 97.30 63.30 Panc-197 88.90 43.40 HEY 97.60 70.50 22 MDA-MB-453 95 94.80 78 JURKAT 88.6088.20 88.10 F147 769-P 44.30 23.40 5 Panc-1 40 11 0 HEY 0 0 0 MDA-MB-45370 50 57 JURKAT 86.30 84 78.70 Percent Growth Inhibition.

Although the above procedures focused on the isolation of activeconstituents from pods of Acacia victoriae, the active constituents mayalso be extracted from roots. In this case, the roots are ground for ½hour and covered with 100% MeOH. The mixture is then filtered anddiluted to 80% MeOH in water. If large amounts of roots are to beextracted, then it may be preferable to extract via percolation asdescribed above. The reason for the differences in these extractionprocedures is that roots are typically extracted fresh while the podsare often dried prior to extraction.

Example 3 Preparative Scale Procedure for Preparing Active Constituentsfrom Fraction UA-BRF-004-DELEP-F094

A modified extraction/separation procedure was used for the scaled-uppreparation of mixtures of active constituents from fractionUA-BRF-004Pod-DELEP-F094 (F094). This procedure was repeated multipletimes, consistently yielding highly active fractions. Typically, 20-25 gof F094 or its equivalent was dissolved in 150-175 ml of 50% MeOH in H₂Owhich was then aspirated onto a column ((26 mm×460 nun) +(70 mm×460 mm),RP-C18, 40 μm, 1200 g, equilibrated with 60% MeOH/H₂O). The fractionswere eluted in steps of 8 L in 60% MeOH/H₂O; 7.5 L 70% MeOH/H₂O; and 2 LMeOH and assigned fraction identifiers as shown in Table 15. FractionF035-B2 contains a mixture of the active components contained in F094,F133-136 (isolated from F093) and F138-147 (isolated from F094) as shownin FIGS. 18A-18F. F094 is an acceptable substitute for F035 with a one-to two-fold decrease in potency and F035-B2 has less potency than F094.

TABLE 15 Isolation of F035-B2. Fraction Volume Collected TotalIdentifier (L) Weight (g) Eluent F237 8 1.8  60% MeOH in water F238 1 8 70% MeOH in water F035-B2 3.5 80  70% MeOH in water F239 3 19  70% MeOHin water F240 2 20 100% MeOH in water

A procedure was designed by the inventors for the further purificationof the active components in Fraction F035-B2 to give fractions havingthe analytical HPLC characteristics of UA-BRF-004-DELEP-F035. Theprocedure is as follows: Additional preparative HPLC is carried out onFraction F035-B2 using 10 micron reversed phase chromatography columnsto elute with step gradient mixtures of acetonitrile and water rangingfrom 26% acetonitrile in water up to 40% acetonitrile in water in 1-2%step gradient fashion, followed by a 100% acetonitrile wash and 100%methanol wash, which will produce a unique breakdown of F035-B2 intoseveral fractions containing the 0 to 20 minute peaks (per standard sixmicron HPLC RP-18 analytical method), which are not contained in theoriginal F035 fraction (FIG. 18A). The remaining fractions obtainedprovide one to three component fractionation of F035. As indicated bythe testing above, fractions F139-F147 are similar to these fractions,with some degree of enhanced anti-tumor in certain cancer cell linesrelative to others. The unique mixture of active components present infraction F035 can be produced in bulk by modifying the originalcomposition of the front end solvents between 16% and 26% acetonitrilein water followed by MPLC purification to produce multi-gram quantitiesof the equivalent of UA-BRF-004-DELEP-F035.

Further improvements to the above extraction procedure, as well as theother extraction procedures disclosed herein, may be realized by usingtri-solvent mixtures of acetonitrile, methanol and water. The percentageranges can be dynamically produced and optimized by anyone familiar withstandard chromatographic techniques. Likewise, bonded phase silicas canbe varied by using a combination of RP systems, including, but notlimited to C-8, CN, dimethyl diol and C-18. In the final steps, evennormal phase silica can be utilized for final purification procedures.

Example 4

Alternative Procedures for Isolating Active Constituents from Acaciavictoriae

Fractions F094 (250 g), F035 (50 mg), and Acacia victoriae ground pods(1 Kg) (i.e. seedpod powder) were obtained as described above. Theanalytical methodology used to analyze fraction F094 and the subsequentfractionation of the F094 are described in this example.

4.1 Analytical Methodology

Several methods involving various C8 and C18 columns under gradient andisocratic conditions were tried to resolve fraction F094. The monitoringincluded both UV at 220 nm and evaporative light scattering detection(ELSD). Better peak resolution was seen with mobile phases containingtrifluoroacetic acid (TFA). The method, called herein as Acacia 257, isdescribed below. This method provides good resolution along with a shortrun time.

The HPLC was equipped with a diode array detector (DAD) or variablewavelength detector and a 4.6×150 mm Inertsil C18 3μ, column (MetaChem).The detector was set at 220 nm.

The following gradient was run.

% H₂O with Time (min) % Acetonitrile 0.1% TFA 0 30 70 36 36 64 42 42 5842.1 30 70 47 30 70

FIG. 25 shows the chromatogram of F094 obtained by this method. F094consists of three groups or families with a number of peaks in eachfamily. Family-1 (8 to 20 min; peaks A-D), Family-2 (22 to 35 min; peaksE-H) and Family-3 (36 to 47 min; peaks I-L). Fraction F035 was alsoanalyzed by this method, and the chromatogram is shown in FIG. 26. Thepeaks of the second family are more abundant in F035 compared to F094where first family peaks are more abundant.

4.2 Fractionations

4.2.1 First Fractionation

The first fractionation focused on the peaks in Family-1. A Symmetry C8semi-prep column (7.8×300 mm, 7μ) (Waters) was employed for this purposewith a gradient elution program as shown below. Seven sub-fraction cutswere made as shown in FIG. 27. The last fraction cut (#2160-007-31)includes all peaks in both Family-2 and Family-3. These fractions aswell as the starting material (F094) were sent for bioassay.

% H₂O with Time (min) % Acetonitrile 0.1% TFA 0.0 27 73 38.0 30 70 42.190 10 48.0 90 10 49.0 27 73 65.0 27 73

4.2.2 Second Fractionation

The separation of the peaks in second family of compounds was the targetof this fractionation. This was achieved with the usage of the same C8semi-prep column. The mobile phase was isocratic 32% acetonitrile inwater containing 0.1% TFA. The chromatographic trace indicating whereseven fraction cuts were made is shown in FIG. 28. The first fractioncut here includes all the peaks in family-1.

4.2.3 Bioassays

The bioassay results of the sub-fractions from the first and secondfractionations are shown Tables 16 and 17 respectively.

TABLE 16 Cytotoxicity in Jurkat cells of sub-fractions from Firstfractionation Cytotoxicity IC₅₀ Fraction No. Weight (mg)* (μg/ml)2160-007-03 2.7 Not active 2160-007-07 1.9 Not active 2160-007-11 1.3Not active 2160-007-15 1.6 Not active 2160-007-19 1.7 1.2 (Peak D1)2160-007-25 2.9 5.7 (Peak D2) 2160-007-31 3.2 1.3 2160-007-34 9.3 0.17(FO94) *These weights are approximate ±20%

TABLE 17 Cytotoxicity in Jurkat cells of sub-fractions from Secondfractionation Cytotoxicity IC₅₀ Fraction No. Weight (mg)* (μg/ml)2160-025-01 7.24 1.2 2160-025-02 4.74 2.8 2160-025-03 3.63 1.02160-025-04 1.37 0.64 (Peak G1) 2160-025-05 2.07 1.56 (Peak G2)2160-025-06 3.64 0.33 2160-007-34 12.09 0.17 (F094) *These weights areapproximate ±20%

Two purified triterpenoid glycosides, namely D1 and G1, were obtainedfrom the Acacia fraction F094. Acid hydrolysis of D1 produces anaglycone.

4.4 Prep Scale Fractionation to Obtain D and G/H Region Peaks

F094 (2.3 g) was fractionated on a HPLC Prep PFP (pentafluorophenyl)column (50×250 mm, 10 μm). The mobile phase was acetonitrile/watercontaining 0.1% trifluoroacetic acid (TFA) run in gradient mode from 27%to 32% acetonitrile over 38 min. As shown in FIG. 29 this methodseparated fractions containing D and G/H peaks. The fraction cuts fromthis prep run were sent for bioassays. The analytical assays of D andG/H are shown in FIGS. 30 and 31. The method used here is Acacia 257 andwhich was described earlier in the same section.

The fraction GM was further purified first on the PFP Prep column toobtain G1 with 68% chromatographic purity, and this material was furtherpurified on a C-18 semi-Prep column to obtain pure G1.

About 100 mg of G/H mixture was loaded on to the same PFP columndescribed before. The following gradient was run.

% H₂O with Time (min) % Acetonitrile 0.1% TFA 0 27 73 1 29 71 40 34 66

Five fractions were collected (G1, G2, G3, H1 and H2). Analytical on G1(FIG. 32) indicated a chromatographic purity of 68%. This fraction wasfurther purified on a semi-prep column.

A YMC C18-Aq column (10×250 mm, 5 μm) was employed. The mobile phase was31% acetonitrile in water with 0.1% TFA. The final G1 product had achromatographic purity of 100%. (FIG. 33).

The fraction D (45% D1) from the PFP Prep column was first fractionatedon a Waters C-18 column (25×100 mm). The mobile phase was 61% methanolin water with 0.1% TFA. The HPLC analysis showed that D1 was 78% pure(FIG. 34) and it contained another peak (named D1.5). A sample of D1with 100% chromatographic purity (FIG. 35) was produced by furtherfractionation of the impure D1 on YMC C18-Aq column with 33%acetonitrile/water with 0.1% TFA as the mobile phase. It was observedthat D1 is more stable in dilute acid solutions than in water at 40° C.Therefore, 0.1% TFA was included in solvents during the purification ofD1.

4.4.1 Bioassays

The bioassays were performed on Jurkat cell lines and the effects ofvarious sub-fractions and pure D1 and G1 are shown Tables 18, 19 and 20respectively. D1 and G1 were tested at two different pH values. Theresults indicated a slightly higher activity at pH 6.5 vs pH 7.5.However, the cell growth was inhibited by about 40% at lower pH values.

4.4.2 Acid hydrolysis of D1

The saponin D1 in ethanol was hydrolyzed with 3N HCl for 3 h at 100° C.The aglycone produced was purified by HPLC. The mass spectral analysisshowed molecular weight of the aglycone to be 652.

TABLE 18 Cytotoxicity fractions from Prep PFP column Cytotoxicity IC₅₀Fraction No. Description Weight (mg)* (μg/ml) 2160-035-22 Peak D 1.70.52 2160-047-01 Peaks G/H 1.24 0.12 2160-047-03 Peaks I/J/K 1.66 0.192160-047-05 Peak L region 1.17 0.18 2160-047-07 Peak M 1.72 0.242160-007-34 F094 0.21

TABLE 19 Cytotoxicity fractions from G/H fractionation Cytotoxicity IC₅₀Fraction No. Weight (mg)* (μg/ml) 2160-53-8-G1 1.87 1.23 2160-53-11-G20.76 2.2 2160-53-14-G3 0.67 4.35 2160-53-17-H1 0.29 6.25 2160-53-20-H20.45 12.8 2160-007-34 (FO94) 0.38

TABLE 20 Cytotoxicity of D1 and G1 at pH 6.5 and 7.5 Cytotoxicity IC₅₀Compound/Extra (μg/ml) ct Weight (mg)* pH 6.5 pH 7.5 2160-69-29 (D1)1.036 1.01 0.98 2160-083-30 (G1) 1.951 0.3 0.49 2160-007-34 (F094) 0.150.22

Example 5 Structures of D1, G1, and B1

5.1 The Structure of D1

D1 is a major component of Acacia victoriae pods. Assays of thiscompound show that it has considerable biological activity.

5.1.1. Whole Molecule D1

D1 was isolated as a colorless amorphous solid isolated from thepartially purified extract F094 obtained using several preparative HPLCseparations as described in the examples above. Its molecular weightfrom MALDI mass spectroscopy is 2104 amu which is the sodium adduct of2081, the true molecular weight. A high resolution FAB mass spectroscopyconfirmed this molecular weight and gave the molecular formula ofC₉₈H₁₅₅NO₄₆. Such a molecule is too large for structure determinationvia spectroscopy alone and so a degradation program was undertaken asoutlined in Scheme 1 shown in FIG. 36. In FIG. 36, D1 is represented bythe structure labeled ‘(1)’.

The proton and carbon NMRs of D1 showed the presence of a triterpene,two monoterpenes and approximately eight sugars (See Table 21 forselected ¹³C-NMR assignments under (1)).

TABLE 21 ¹³C NMR (MeOH-d4) assignments of D1(1), G1(14), B1(21),Aglycone (2) and Acacic acid (3). (The numbers in brackets i.e., 1, 14,21, 2 and 3, refer to structures of D1, G1, B1, Aglycone and Acacicacid, depicted in FIG. 36, FIG. 37 and FIG. 38 respectively.) Carbon No.(1) (14) (21) (2) in DMSO-d6 (3) Triterpene Part 1 36.13 36.13 36.1336.07 38.90 2 27.15 27.15 27.15 29.28 28.03 3 89.86 89.84 76.78 77.94 440.09 39.85 39.71 39.28 5 57.08 54.84 55.78 6 19.54 18.03 18.71 7 34.5934.59 34.58 34.27 33.51 8 40.82 40.09 40.82 39.79 9 48.08 46.11 47.1510  37.94 37.94 36.59 37.31 11  24.29 24.54 24.49 26.97 23.77 12  124.04124.04 124.09 122.04 122.61 13  143.70 143.7 143.68 142.61 144.29 14 42.64 42.63 42.01 15  36.20 36.39 36.51 35.74 16  74.26 72.41 74.22 17 52.29 49.70 51.67 18  41.64 41.60 40.97 19  48.67 48.3 46.85 48.42 20 35.88 35.95 36.64 21  78.61 76.78 73.32 22  39.86 41.7 41.94 38.07 41.9723  28.62 28.61 28.65 26.60 28.65 24  17.12 17.11 17.11 16.06 15.55 25 16.22 16.22 16.25 15.19 16.47 26  17.73 17.72 18.07 16.78 17.43 27 27.40 27.32 27.40 28.24 27.11 28  173.39 175.34 175.39 176.64 179.14 29 29.41 29.43 29.41 28.77 29.97 30  19.42 19.42 19.53 18.65 18.26 OuterMonoterpene 1 168.69 168.68 168.74 2 132.92 132.92 132.82 3 148.48148.02 4 24.49 24.58 24.56 5 41.95 41.33 40.83 6 81.01 81.0 7 145.93144.01 8 112.53 112.44 112.53 9 16.75 16.7 16.74 10  56.51 12.49 InnerMonoterpene 1 168.17 169.01 168.19 164.0 2 132.49 128.52 132.49 135.20 3148.03 145.95 137.05 4 24.29 24.29 24.30 22.86 5 41.33 39.86 39.73 76.036 73.61 129.41 7 144.03 144.43 119.80 8 116.0 116.0 115.33 11.86 9 23.7623.7 24.21 12.81 10  56.62 56.61 64.28

5.1.2. Vigorous Acid Hydrolysis of D1

Hydrolysis of D1 in 3N HCl at 100° C. for 2 hrs. produced “D1 aglycone”,depicted as (2) in FIG. 36, which was shown by mass spectroscopy to havea molecular weight of 652. The NMR of D1 aglycone showed the presence ofa triterpene and a modified monoterpene but no sugars. This material wasfurther degraded by saponification (1.3N NaOH at 100° C. for 30 min. inMeOH) from which the following were isolated:

5.1.2.a. Triterpene The C-13 NMR of this material was identical withthat reported previously for acacic acid (see FIG. 36 structure depictedby (3), and see Table 21 for ¹³C-NMR assignments under (3)) and itsmolecular weight by mass spectroscopy at 488 is consistent with thisstructure.

5.1.2.b. Cyclized Monoterpene The molecular weight and NMR of thiscompound indicated the presence of a carboxylic acid, two methyl groupsattached to a double bond and two vinyl protons leading to the pyranestructure indicated. While this structural unit Structure depicted by(4) FIG. 36, was also present in “D1 aglycone”, it was not present inthe parent D 1. The D1 contains the acyclic monoterpene, depicted asstructure (5) in FIG. 36, and this structure undergoes cyclizationduring the acid hydrolysis as shown below:

These structures along with the original molecular weight and spectralcharacteristics of D1 fit well with the structure of D1 aglyconedepicted in FIG. 36 by the structure labeled (2). See Table 21 forselected ¹³C-NMR assignments under (2).

5.1.3. Mild Saponification of D1

When D1 was treated with 0.5N NH₄OH at room temperature for 1 hour therewas complete conversion into two new compounds.

5.1.3.a. Monoterpene This molecule had a molecular weight of 200 and NMRwhich indicated that it possessed an acyclic monoterpene structure,supporting the suspected degradation. This structure is depicted in FIG.36 and is labeled (5).

5.1.3.b. Triterpene Monoterpene Oligosaccharide This compound is morepolar than D1 and its NMR is consistent with it containing acacic acid,one monoterpene and several monosaccharides. This structure is depictedin FIG. 36 and is labeled (6).

5.1.4. Sugar Analysis of D1

A vigorous acid hydrolysis of D1 (2N HCl at 100° C. for 2 hours)followed by derivatization (trimethylsilyl ethers) and GC/MS analysisconfirmed the presence of eight sugar residues in the original molecule:arabinose, rhamnose, fucose, xylose, 6-deoxyglucose (i.e. quinovose),N-acetyl glucosamine and two molecules of glucose.

5.1.5. More Aggressive Saponification of the Triterpene MonoterpeneOligosaccharide

When the triterpene monoterpene oligosaccharide was subjected to 0.3NNaOH for 1 hour at 60° C. three compounds were formed:

5.1.5.a. Oligosaccharide Isolation and analysis of this very polarfragment suggested that it was an oligosaccharide. Sugar analysisperformed by acid hydrolysis (2N HCl at 100° C. for 2 hours) and GC/MSanalysis of the trimethylsilyl ethers of the monosaccharides confirmedthat the oligosaccharide was a tetrasaccharide made up of two moleculesof glucose and one each of arabinose and rhamnose.

5.1.5.b. Monoterpene Glycoside This material has NMR's consistent withstructure (8) depicted in FIG. 36. Acid hydrolysis (2N HCl at 100° C.for 2 hours) of this compound led to the identification of the sugar as6-deoxy glucose. Treatment of this monoterpene glycoside withβ-glucosidase gave the monoterpene with the structure depicted by (9) inFIG. 36, which has an NMR consistent withtrans-2-hydroxymethyl-6-hydroxy-6-methyl-2,7-octadienoic acid.Hydrolysis of this linkage with a “beta”-glucosidase indicates that thelinkage between these two groups is a beta linkage.

5.1.5.c. Triterpene Glycoside This compound has a molecular weight of951 and NMR's which is consistent with the acacic acid lactonecontaining a trisaccharide at the C-3 position depicted by structure (10b) in FIG. 36. Acid hydrolysis (2N HCl at 100° C. for 2 hours) of thiscompound allowed the identification of its constituent sugars asN-acetyl glucosamine, fucose, and xylose by GC/MS as trimethyl silylderivatives. This molecule was observed in both the open acid/alcohol,which is depicted in FIG. 36 by the structure labeled (10 a), and theclosed lactone form, which is depicted in FIG. 36 by the structurelabeled (10 b).

Sugar analysis and molecular weight of the fragments as compared withthose in the whole molecule D1 confirmed that all portions of D1 wereaccounted for in fragments depicted by structures labeled (5), (7), (8),and (10 a) in FIG. 36.

5.1.6. Mild Acid hydrolysis of D1

Mild acid hydrolysis of D1(1N HCl for 16 hrs at 25° C.) allowed theformation of two new molecules:

5.1.6.a. Monoterpene Sugar The molecular weight, NMR spectra, and sugaranalysis were consistent with a monoterpene-6-deoxyglucose. Thestructure of this molecule is depicted in FIG. 36 by the structurelabeled (11).

5.1.6.b. Triterpene-Monoterpene-Glycoside The second molecule wasidentified to be a triterpene-monoterpene-glycoside and the structure ofthis molecule is depicted in FIG. 36 by the structure labeled (12).

5.1.7. The Attachment of Subgroups within D1

NMR studies indicate that the carboxylic acid of the outer monoterpeneis esterified to C-4 of 6-deoxyglucose (quinovose). NMR and hydrolysisstudies have shown that the anomeric carbon of the quinovose is attachedto the C-6 hydroxy group of the inner monoterpene. The stereochemistryat the anomeric carbon of quinovose indicate a “beta” linkage.

Hydrolysis (2N HCl for 2 hrs at 100° C.) and sugar isomerization studiesindicate that the sugars in the tetrasaccharide are two molecules ofglucose, and one molecule each of rhamnose and arabinose. The unit isdirectly esterified to the C-28 carboxylic acid of the triterpene asshown in FIG. 39. Iron trap mass spectroscopy studies indicate that thetetrasaccharide structure has two glucose and one arabinose attached toa central rhamnose as shown in FIG. 39. The linkage of these sugars oneto another is still unknown.

NMR studies indicate that N-acetyl glucosamine (NAG) is attacheddirectly to the C-3 carbon of the triterpene. The remainder of thesequence of the sugars is fucose in the middle and xylose on the end byLC/MS studies of partial hydrolysis (1N HCl for 1 hr at 60° C. in 50%MeOH). The linkage of these sugars one to another is still unknown.

5.1.8. Elliptoside E

D1 contains a triterpene and two monoterpenes commonly found in sapohinsreported from other species including other Acacia. Although thestructure of D1 is similar to elliptoside E, (FIG. 24), reported fromArchidendron ellipticum, (Beutler et al., 1997). In the presentinvention, the specific rotation of D1 has been determined to be[α]_(D)=−30.0° which is different than the reported value forelliptoside E at −24.3° .

Elliptoside E, described in Beutler et al. (1997_, and D1 have differentHPLC retention times (D1-15.2 min., elliptoside E—12.5 min.). Therefore,these two molecules must differ in some manner such as the specificattachment of their subunits or from the presence of optical orstructural isomers.

The inventors observed that the specific rotation of the innermonoterpene, depicted by structure (9) in FIG. 36, is +11.2° in MeOH and+16° in chloroform. This same fragment in elliptoside E was reported tobe −9.1° in chloroform. Furthermore, the only chiral center of the innermonoterpene of D1 was determined to have an “S” configuration which isopposite to that found in elliptoside E. The specific rotation of theouter monoterpene of D1 is being sought at this time. Furthermore,proton NMR shows that the monoterpene double bonds in D1 are “trans”whereas the monoterpene double bonds are “cis” in elliptoside E as shownin Beutler et al., 1997. These two features constitutes the firststructural differences found between D1 and elliptoside E. Enzymaticcatalytic hydrolysis of specific sugars has shown that the arrangementsof sugars is the same as in elliptoside E.

5.2. The Structure of G1

Biological assays of this material shows that G1 is more biologicallyactive than D1.

5.2.1. Whole Molecule G1(14)

The second structure determined in the present invention was G1. It wasalso isolated from F094 by prep HPLC but in low compound recovery. G1 isslightly less polar than D1. The molecular weight by MALDI massspectroscopy indicates a molecular weight of 2065 which is 16 amu lessthan D1. Specific rotation of G1 was found to be −26.9° (MeOH). Theproton NMR shows that G1 is also a saponin, very similar to D1 andindicates that it only differs from D1 by having one less oxygen in theouter monoterpene which is nowtrans-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid. See FIG. 37,structure labeled (14), and Table 21 for selected ¹³C-NMR assignmentsunder (14). G1 was degraded as shown in Scheme 2, FIG. 37.

5.2.2. Mild Saponification of G1

When G1 was treated with 0.5 N NH₄OH at room temperature for even a fewminutes there is complete conversion into the more polar mildsaponification product and a monoterpene.

5.2.2.a. Monoterpene The molecular weight and NMR of this materialindicates that it possesses a methyl group at the C-2 position where ahydroxymethyl had been in. This is depicted in FIG. 37 by the structurelabeled (15).

5.2.2.b. Triterpene Monoterpene Oligosaccharide The NMR of this compoundindicates that it was identical by HPLC retention time and by proton NMRwith the structure labeled (16) depicted in FIG. 37, which is similar tothe structure labeled (6) in FIG. 36 made from D1 and that it containsan acacic acid, one monoterpene and eight monosaccharides as was seen inD1. The similarity of (16) with (6) indicates a similar stereochemistryseen in D1 inner monoterpene.

5.2.3. Sugar Analysis of G1

A vigorous acid hydrolysis of G1 (2N HCl at 100° C. for 2 hours)produced the same monosaccharide units as were present in D1: arabinose,rhamnose, fucose, xylose, 6-deoxyglucose, N-acetyl glucosamine and twomolecules of glucose.

5.2.4. Acid Hydrolysis of G1

An acid hydrolysis of the mild saponification product allowed theisolation of three molecules in a manner as in D1. NMR and sugaranalyses (2N HCl at 100° C. for 2 hours) were performed on each. This isdepicted in FIG. 37 by the structure labeled (16).

5.2.4.a. Oligosaccharide contained two molecules of glucose and one eachof arabinose and rhamnose and is depicted in FIG. 37 by the structurelabeled (17).

5.2.4.b. Monoterpene Glycoside contained an acyclic monoterpene(depicted in FIG. 37 by the structure labeled (5)), and 6-deoxyglucoseand the whole structure is depicted in FIG. 37 by the structure labeled(18).

5.2.4.c. Triterpene Glycoside contained acacic acid and one moleculeeach of N-acetyl glucosamine, fucose, and xylose. The sugars in thesefragments are arranged in the same order as in D1. This structure isdepicted in FIG. 37 by the structure labeled (19).

5.2.5. Elliptoside A

G1 has the same terpene content and sugars as elliptoside A (see FIG. 24and Beutler, 1997). However, elliptoside A was found to have a markedlydifferent HPLC retention time (G1 −29.09 min. and elliptoside A −26.04min.), which indicates that the two molecules must differ in some mannersuch as the specific attachment of their subunits or from the presenceof optical isomers or both. A comparison of the proton and carbon NMRspectra of G1 and elliptoside A also show differences in chemicalshifts. It is contemplated that the specific rotations of the inner andouter monoterpenes of these compounds may also differ. FIG. 37 structure(14) represents the structure of G1.

5.3. The Structure of B1

Bioactivity data indicates that Bi is much less active than D1 or G1.

5.3.1. Whole Molecule B1(21)

The isolation of B1 was accomplished by plant extraction and C-18 flashchromatography followed by C-18 prep and semi-prep chromatography. TheNMR of B1 indicates the sametriterpene/monoterpene/quinovose/monoterpene structure as has been seenthroughout this saponin family. The NMR also indicates the presence offour deoxy sugars and one N-acetyl group, which indicates that thismolecule must differ from D1 in its sugar portions. See Table 21 forspecific ¹³C-NMR assignments under (21). This molecule was degraded asshown in FIG. 38.

5.3.2. Sugar Analysis of B1

NMR data indicate the presence of more than one copy of one of the6-deoxy methyl sugars (i.e. fucose, rhamnose, 6-deoxyglucose). Sugaranalysis of the total molecule after hydrolysis (2N HCl at 100° C. for 2hours) indicates that nine sugars are present: one molecule each offucose, arabinose, xylose, quinovose, and glucosamine and two moleculesof glucose and rhamnose. Glucosamine, the remnant of an N-acetylglucosamine, is present in the original molecule. The structure of B1 isdepicted in FIG. 38, structure (21).

5.3.3. Mild Saponification of B1

When B 1 was treated with 0.5 N NH₄OH at room temperature for even a fewminutes there is complete conversion into a more polar compound, themild saponification product, and a monoterpene.

5.3.3.a. Monoterpene The molecular weight and NMR of this materialindicates that it has the same structure as the monoterpene from D1,depicted in FIG. 37 by the structure labeled (5). This is depicted inFIG. 38 by the structure labeled (22).

5.33.b. Triterpene Monoterpene Oligosaccharide The NMR of this compoundindicates that it contains acacic acid, one monoterpene and severalmonosaccharides. This is depicted in FIG. 38 by the structure labeled(23).

More Aggressive Saponification of the Triterpene MonoterpeneOligosaccharide

A more aggressive saponification (0.3N NaOH at 60° C. for 1 hour) of themild saponification product allowed the isolation of three molecules ina similar manner as before in D1 and G1. Sugar analyses and NMR datawere obtained for each.

5.3.4.a. Oligosaccharide contained glucose, arabinose and two moleculesof rhamnose. This is depicted in FIG. 38 by the structure labeled (24).

5.3.4.b. Monoterpene Glycoside contained 6-deoxyglucose and amonoterpene. This is depicted in FIG. 38 by the structure labeled (25).

5.3.4.c. Triterpene Glycoside contained acacic acid with atetrasaccharide attached at the C-3 position. The tetrasaccharide iscomposed of one molecule each of N-acetyl glucosamine, fucose, glucose,and xylose. This is depicted in FIG. 38 by the structure labeled (26).

Example 6 De-Esterification of the Triterpene Compounds of the Invention

F094 was de-esterified and the de-esterified products bioassayed toelucidate the active components. 1.00 g of F094 was dissolved in 100 mlof H₂O, followed by addition of 1 g of KOH and refluxing for 1.5 hrs.The solution was allowed to cool to room temperature and its pH adjustedto 7 with 1N HCl and then washed with hexanes (2×50 ml). The aqueoussolution was then subjected to further stepwise extractions to yieldfractions 159-162. For example, the solution was initially extractedwith n-butanol (2×50 ml) to yield 0.127 g of organic soluble solid (F159) after drying in vacuo. The aqueous layer was acidified to pH 5 with1 N HCl and extracted with EtOAc (2×50 ml) to yield 0.397 g of an EtOAcsoluble solid (F160), then n-butanol (2×50 L) to yield 0.338 g of solid(F 161) after removal of the organic solvents. The aqueous layer wasfinally neutralized to pH 7 with 1N NaOH. 1.808 g of solid (F162) wasisolated from the final aqueous layer.

Bioassays revealed that the de-esterified products had little or noactivity. F 159-162 were bioassayed for cytotoxicity against 769-P,Panc-1, HEY, MDA-MB-453 and Jurkat cell lines. The only activity foundwas for F161, which exhibited a cytotoxicity of 1.6% against MDA-MB-453at 50 μg/ml and for F159 which exhibited cytotoxicities of 15.50%,6.60%, and 3.80% against Jurkat cells at 50 μg/ml, 25 μg/ml, and 12.5μg/ml, respectively. These results indicate that the ester side chain isnecessary for bioactivity. It is believed that the ester side chain ofthe compounds of the invention exhibits anti-tumor activity and/or worksin concert with the triterpene skeleton of the compounds of theinvention to produce the potent anti-tumor activity exhibited.

Example 7 Sugar Hydrolysis of the Compounds of the Invention

The sugars contained in F094 were also hydrolyzed to aid in thecharacterization of the active components. 12 g of F094 was dissolved in400 ml 2N H₂SO₄ and refluxed for 75 min during which time an insolublematerial formed. The solution was cooled and filtered through sinteredglass. The residue was washed with water, yielding 4.8 g of anaglycone(s) (F 191), as determined by TLC analysis. The dark amberfiltrate was neutralized with KOH or NaOH. A white precipitate wasformed and collected. The addition of isopropanol to the amber filtratecaused a second white precipitate. The solvent was removed in vacuo andthe solvent re-suspended in MeOH which resulted in the formation of awhite precipitate which probably corresponded to sulfate salts asdetermined by a flame test. The almost clear filtrate dried in vacuo andthe residue acetylated and analyzed by HPLC to contain a mixture ofsugars as shown in FIGS. 17A and 17B. This mixture probably contains atleast 5 saccharides. These sugars may be further characterized byisolation of the TMS derivatives for GC-MS characterization; paperchromatography; isolation of the benzyl derivatives for HPLC separationor DEPT NMR; or C¹³ NMR as fully described herein above. By standard 1-Dand 2-D cellulose, paper and normal phase TLC, the main sugars have beenidentified as glucose, xylose, rhamnose and arabinose along with someminor additional sugar and a strong potential for an amino glycoside,especially an acetamido substituted sugar.

The number of different sugars probably explains the complicated HPLCspectra, which show the presence of dozens of closely-related compounds.In particular, some active constituents appear to be glycosylated at twodifferent sites (an alcoholic and a carboxylic acid site). Combinationsof six sugars attached to two sites would thus yield large numbers ofclosely-related compounds which would be hard to separate.

Alternatively, milder hydrolysis conditions run with 100% ethanol(azeotrope with 5% water) and 0.1 to 2.0 N H₂SO₄ (the remaining work-upis the same) under mild heating to the point of reflux, but not vigorousreflux, generates a similar mixture of aglycones. Some components aremissing which indicates that some isomerization takes place under thestronger conditions.

The aglycone(s) F191 (1 g) was then methylated by refluxing 5-7 hourswith methyl iodide (1 ml) and anhydrous K₂CO₃ (1 g) in anhydrous acetone(10 ml). This resulted in 0.315 g of an insoluble material and 0.54 g ofmethyl esters denoted F197. 500 mg of F197 was further separated by MPLCemploying a 15×460 mm column (45 g SiO₂, 15-25 μm) where the sample waspre-adsorbed onto 1.5 g SiO₂, 15-25 μm. The compounds were eluted with790 ml 7% isopropyl alcohol (IPA) in hexanes (subfractions 1-10), 470 ml10% IPA in hexanes (subfractions 1-14), 275 ml 20% IPA/hexanes(subfractions 14-15), 200 ml dichloromethane, and 100 ml DCM/MeOH (1:1),in accordance with Table 22.

Bioassays of F191 and F197 yielded cytotoxicities for ovarian cancercells (line HEY), renal cancer cells (line 769-P), pancreatic cancercells (line Panc-1), Jurkat T-leukemic cells, and MDA-MB-453 breastcancer cells at corresponding dosages as indicated in Table 23.

TABLE 22 Fractionation of F197 to Fractions F198 to F208. SubfractionsTotal Fraction Collected Weight Identifier (volume (ml)) (mg) CommentsF198 1 (100) 14 F199 2-3 (120) 126 Further fractionated to F209-214.F200 4 (40) 8 F201 5-6 (110) 86 Further fractionated to F215-219. F2027-8 (170) 37 F203 9-10 (250) 17 F204 11 (100) 5 F205 12 (150) 38 F206 13(150) 10 F207 14-15 (345) 86 F208 16-18 (300) 105

TABLE 23 Bioassay of Fractions F191 and F197. 50 μg/ml 25 μg/ml 12.5μg/ml F191 769-P 82.3 56.3 33.7 Panc-1 90 64 40.3 HEY 94.5 71.6 0MDA-MB-453 53.5 22.3 7.3 JURKAT 69.6 68.6 45.3 F197 769-P 84.3 61.1 40.5Panc-1 93.5 84.4 53.8 HEY 94.4 94.7 62.1 MDA-MB-453 76.8 79.2 68.8JURKAT 70.2 70.6 56.9

F199 (116 mg) was further fractionated by the same column used tofractionate F191 and eluted in 100 ml 2% IPA/hexanes, 525 ml 4%IPA/hexanes, and 250 ml 10% IPA/hexanes according to Table 24.

TABLE 24 Fractionation of F199 to Fractions F209 to F214. SubfractionsTotal Fraction Collected Weight Identifier (volume (ml)) (mg) CommentsF209 1-7 (225) 5 F210 8 (20) 1 F211 9 (20) 2 F212 10-14 (140) 90 Furtherfractionated to F220-F228. F213 15-17 (220) 17 F214 18 (250) 10

F212 (85 mg) was further fractionated by a Waters Prep LC 4000 HPLC on a22×500 mm column (Alltech Econosil C18, 10 μm, equilibrated with 75%ACN/water) and eluted in 80% ACN/water and washed with ACN at a rate of40 ml/min, for a detection limit of 220 nm and subfractions collectedevery 30 sec (20 ml) according to Table 25.

F223 was initially purified as its methyl ester derivative to giveC-191. C-191 was subjected to a classical acetylation procedure.Specifically, C-191 (47 mg) was stirred overnight at room temperature ina 2:1 mixture of acetic anhydride and pyridine. The reaction wasquenched with water and the solution partitioned with diethyl ether and5N HCl. The organic layer was then washed until neutral, roto-evaporatedand the residue subjected to PTLC—one 20 cm by 20 cm preparative plateeluted with 90:10 hexane:isopropyl alcohol, followed by subsequentPTLC's eluted with dichloromethane:methanol (98:2) to C-191 acetate(F229, which later was given the number C-194).

TABLE 25 Fractionation of F212 to Fractions F220 to F228. SubfractionsTotal Fraction Collected Weight Identifier (volume (ml)) (mg) CommentsF220 A (940) 12 F221  1-24 1 F222 25-27 3 F223 28-38 55 “C191” -targeted for characterization as its acetylated derivative by ¹³C- and¹H-DEPT NMR, HPLC, RP18 TLC and MS. F224 39-41 3 F225 42-54 4 F226 55-741 F227  75-102 5 F228 103 2 ACN wash

F201 (85 mg) was also further fractionated by MPLC by a similar columnas used to fractionate F199 and eluted and collected in 2% IPA/hexanes(120 ml), 4% IPA/hexanes (330 ml), 7% IPA/hexanes (460 ml), 20%IPA/hexanes (150 ml), DCM (50 ml), and DCM/MeOH (1:1) (70 ml) accordingto Table 26.

TABLE 26 Fractionation of F201 to Fractions F215 to F219. SubfractionsTotal Fraction Collected Weight Identifier (volume (ml)) (mg) CommentsF215 1-5 (510) 3 F216 6-10 (175) 54 “Aglyc II methyl ester” - alsotargeted for characterization. F217 11-14 (225) 4 F218 15 (150) 14 F21916 (120) 10

Example 8 Biological Characteristics of Active Triterpenes of theInvention

Angiogenesis or neovascularization is a process by which cells arerecruited by factor(s) produced by a tumor to provide the tumor with anourishing vascular system. Inhibiting angiogenesis inhibits tumorexpansion by limiting blood supply to the tumor. This function wasexamined using a bovine capillary endothelial cell proliferation assayon cells treated with Fraction 35 (UA-BRF-004-DELEP-F035). The assay wascarried out as follows: bovine capillary endothelial cells were obtainedand grown using standard procedures (Folkman et al., 1979). The cellswere washed with PBS and dispersed in a 0.05% trypsin solution. A cellsuspension (25,000 cells/ml) was made with DMEM+10% BCS+1% GPS, platedonto gelatinized 24 well culture plates (0.5 ml/well) and the suspensionincubated for 24 h at 37° C. The media was replaced with 0.25 ml ofDMEM+5% BCS+1% GPS and different concentrations of UA-BRF-004-DELEP-F035applied. After a 20 min incubation, media and bFGF were added to obtaina final volume of 0.5 ml DMEM+5% BCS+1% GPS+1 ng/ml bFGF. After 72 h thecells were dispersed in trypsin, resuspended in Hematall (FischerScientific, Pittsburg, Pa.) and counted by coulter counter (O'Reilly etal., 1997).

The results of the assay demonstrated significant inhibition ofendothelial cell proliferation with or without basic fibroblast growthfactor (FIG. 5). These results demonstrate that the active components ofthe plant extract are potent inhibitors of endothelial cellproliferation, which is often a predictor of in vivo suppression ofangiogenesis. In addition, the fraction had no effect on migration ofcapillary endothelial cell, suggesting lack of toxicity to normal cells(FIG. 6).

A common problem encountered with steroidal saponins (i.e. digitonin,and the genin-diosgenin from yams) is the lysis of red blood cells.Using a simple culture tube blood assay there was very little detectablelysis following treatment with 1 mg/ml of UA-BRF-004-DELEP-F035.Alternatively, treatment with 10 to 25 dμg/ml digitonin resulted in 100%lysis in the culture tube blood assay.

Next, in order to further study the mechanism by which the activecomponents inhibited tumor cells, the TNF-alpha induced activation ofthe transcription factor NF-κB was analyzed in Jurkat cells (3×10⁶)which had been treated with 1-2 μg/ml of UA-BRF-004-DELEP-F035 andUA-BRF-004Pod-DELEP-F094. The study was carried out as follows: Jurkatcells were pretreated with 1-2 μg/ml of F035 or F094 for 15 h at 37° C.Cells were harvested and resuspended in 1 ml RPMI and treated with 100pM of TNF-alpha for 30 min at 37° C. After TNF-alpha treatment, nuclearextracts were prepared according to Schreiber et al. (1989). Briefly,the cells were washed with ice cold PBS and suspended in 0.4 ml of lysisbuffer.(10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM EDTA, 0.1 mMEGTA, 1 mM dithiothreitol, 0.5 mM PMSF, 2 μg/ml of leupeptin, 2 μg/ml ofaprotinin and 0.5 mg/ml benzamidine). The cells were allowed to sit onice for 15 min and 25 μl of 10% Nonidet-40 was added to the cells. Thetubes were mixed on the vortex and microcentrifuged for 30 s. Thenuclear pellet was resuspended in 25 μl of ice cold nuclear extractionbuffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMdithiothreitol, 1 mM PMSF, 2 μg/ml leupeptin, 2.0 μg/ml aprotinin and0.5 mg/ml benzamidine) and tubes were incubated on ice with intermittentagitation. The nuclear extract was microcentrifuged for 5 runs at 4° C.and supernatants were stored at −70° C.

An electrophoretic mobility shift assay was performed by incubating thenuclear extracts (7 μg of protein) with ³²P-labeled NF-κBoligonucleotides (SEQ ID NO:1; NF-κB consensus oligonucleotide; SantaCruz Biotechnology) in presence of 0.5 μg of poly di-dc in a bindingbuffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1%Nonidet P-40, 5% glycerol and 50 mM NaCl for 20 min at 37° C.) (Nabeland Baltimore, 1987; Collart et al., 1990; Hassanain et al., 1993). TheDNA-protein complex formed was separated from free oligonucleotide on 5%native polyacrylamide gel using buffer containing 50 mM Tris, 200 mMglycine and 1 mM EDTA. The gel was fixed in 10% acetic acid and driedand the bands were visualized using autoradiography with intensifyingscreen at −70° C.

The results of the EMSA demonstrate that in untreated cells there is alow basal level of NF-κB which is activated by TNF (FIG. 20, Lanes 1 and2). Pretreatment of cells with 1 μg/ml of F035 or F094 followed by TNFactivation (FIG. 20, Lanes 4 and 8) resulted in no inhibition of NF-κBactivation. When cells were treated with 2 μg/ml ofUA-BRF-004-DELEP-F035 or UA-BRF-004Pod-DELEP-F094 (FIG. 20, Lanes 6 and10), marked inhibition of TNF activated NF-κB was observed. The resultsof this study indicate that both F035 and F094 were capable of inducinga strong anti-inflammatory response. In addition to indicating theactive triterpene compounds as potential anti-inflammatory compounds,the results are particularly significant given the increasing evidencedemonstrating the central role that inflammation plays in carcinogenesis(Sieweke et al., 1990; Prehn, 1997; Schuh et al., 1990).

Example 9 Studies on Signal Transduction Pathway F035

In order to further elucidate the molecular targets of the activecomponents of the Acacia victoriae plant extract, a study was conductedon the effect of F035 on phosphatidylinositol 3-kinase (PI3-kinase)activity, as well on AKT (protein kinase B, a serine-threonine kinase)activity, a downstream effector of PI3-kinase. PI3-kinase is an enzymewhich is implicated in growth factor signal transduction by associatingwith receptor and non-receptor tyrosine kinases. There are two knownPI3-kinase inhibitors: wortmannin, a fungal metabolite, and LY294002, asynthetic compound which is structurally similar to the plantbioflavonoid quercetin.

The assay was carried out as follows: Jurkat cells (1×10⁷) were starvedovernight and exposed to different concentrations (1-8 μg/ml dependingupon the cells line) of F035 for various times (2-16 h) at 37° C. Afterdifferent time points, the cells were collected and washed with PBS at2000 rpm for 10 min. The cells were lysed in 1% NP-40 lysis buffer for30 min at 4° C. and the lysates isolated by centrifugation for 5 min at15,000 rpm at 4° C. In order to conduct immunoprecipitation ofPI3-kinase, 5 μl of rabbit anti-p85 antibody (tyrosine kinase receptoradapter protein; Upstate Biotechnology Inc.) was incubated with 1 ml ofcell lysate for 90 min at 4° C. The immune complexes were isolated on100 μl of 20% Protein A-Sepharose beads for an additional 90 min at 4 °C. The immunoprecipitates were washed sequentially in a) PBS, 100 mMNa3VO4, 1% Triton -X100; b) 100 mM Tris, pH 7.6, 0.5 LiCl, 100 mMNa3VO4; c) 100 mM Tris, pH 7.6, 100 mM NaCl, 1 mM EDTA, 100 mM Na3VO4;and d) 20 mM Hepes pH 7.5, 50 mM NaCl, 5 mM EDTA, 30 mM NaPPi, 200 mMNa3VO4, 1 mM PMSF, 0.03% Triton X-100. Immunoprecipitates were thenresuspended in 30 μl of kinase reaction buffer (33 mM Tris, pH 7.6, 125mM NaCl, 15 mM MgCl₂, 200 mM of adenosine, 20 mM ATP, 30 μCi [g-32P}ATP). Phosphatidyle inositol (PI; 50 μl) was dried under nitrogen gasand resuspended in 20 mM HEPES, pH 7.5 at 2 mg/ml and sonicated on icefor 10 min. The PI3-kinase reaction was initiated by addition of 10 μlof the PI suspension and 10 μl of gamma-ATP. The reaction was allowed toproceed for 30 min at room temp, followed by termination of the reactionby addition of 100 p.1 of 1N HCl. Lipids were extracted with 600 μlchloroform: methanol (1:1) and resolved on silica gels (G60) bythin-layer chromatography (TLC) in chloroform : methanol: NH₄OH:H₂0(60:47:2:11.3). Radio labeled phosphatidylinositol phosphate wasvisualized by autoradiography and inhibition was quantitated by stormsystem (Okada et al., 1994; Vlahos et al., 1994). The results (FIG. 21)indicate that 2 and 6 hours post-treatment with F035 (4 μg/ml) there wasan inhibition of PI3-kinase activity. Similarly, when cells were exposedto 2 μg/ml of F035 for 15 h, a 95% inhibition was observed, similar towortmannin (a fungal metabolite and known inhibitor of PI3-kinase) inJurkat cells.

Next, the effect of F035 on AKT, a downstream effector of PI3-kinase,was studied. AKT, also known as protein kinase B, is a cellularhomologue of viral oncogene v- AKT and is activated by number of growthfactors and functions in a pathway involving PI3-K activation, which issensitive to wortmannin. AKT codes for serine-threonine protein kinase,which has been shown to be amplified in 12.1% of ovarian carcinomas and2.8% of breast cancers. AKT is involved in an anti-apoptotic pathwaythrough phosphorylation of Bad, an anti-apoptotic molecule. Ovariancancer patients with AKT alterations appear to have poor prognosis(Bellacosa et al., 1995). AKT has been shown to actively blockapoptosis, partly by activation of p70S6 kinase (Kennedy et al., 1997).p70S6 kinase is a mitogen activated serine-threonine protein kinaserequired for cell growth and G1 cell cycle progression (Chou and Blenis,1996). The activity of p70S6 kinase is controlled by multiplephosphorylation events located within catalytic and pseudosubstrateregion (Cheatham et al., 1995; Weng et al., 1995).

The effect of F035 on phosphorylation of AKT was analyzed as follows.Jurkat cells (5×10⁶) were serum starved and exposed to F035 for 15 h and2 h with wortmannin at 37° C. The cells were either induced with cd3XL(cd3 crosslink) or left uninduced for 10 min at 37° C. and lysed in AKTlysis buffer and the proteins were resolved on 8% SDS-PAGE gels andanalyzed by western-ECL using phospho-specific AKT (Ser 473; New EnglandBiolabs) and total AKT antibody. An assay of the effect of F035 on p70S6kinase can be carried out similarly, but using a Phosphoplus p70S6kinase antibody kit (New England Biolabs) for analysis of p70S6 kinase(Ser 411, thr421/ser424) phosphorylation. The results of the AKTanalysis (FIG. 22), demonstrated that cd3 crosslink induces phospho AKTslightly. Post treatment of cells with 1 and 2 μg/ml of F035 caused amarked inhibition of AKT phosphorylation (active AKT), which is similarto a 2 h treatment of cells with 1 μM of wortmannin. There was, however,no change in the expression of total AKT. Similar inhibition of AKTphosphorylation was also demonstrated using ovarian cancer cells OVCAR-3and C-2 (HEY variant), and with Jurkat cells treated with 2-4 μg/ml ofF094. These findings demonstrate that F035 inhibits the phosphorylationof AKT in Jurkat cells and ovarian cancer cells. This is significantgiven that the PI3 kinase/ AKT signaling pathway has been shown todeliver an anti-apoptotic signal (Kennedy et al., 1997). The resultssuggest F035 and F094 is mediating apoptosis of tumor cells through thesuppression of the PI3-K signaling pathway.

Example 10 Cell-Cycle Analysis and Annexin-V Binding Assay To DetectApoptosis

In order to further characterize the mechanism of growth inhibition andcytotoxicity of the active compounds of the plant extract, approximately1×10⁶ OVCAR-3 tumor cells were plated in 60 mm³ dishes, treated withvarious concentrations of UA-BRF-004-DELEP-F035, and incubated for 18-24hours at 37° C. The cells were harvested, washed with PBS twice andresuspended at 1×10⁶ cells/ml. Paraformaldehyde (1% final concentration)was added, drop-by-drop, to cells being gently vortexed. The cells wereagain washed with PBS after a 15 minute incubation on ice, and thepellet was resuspended in 70% ice cold ethanol and incubated at −20° C.overnight. Finally, the ethanol was washed off once with PBS and thecells were resuspended in 10 μg/ml of propidium iodide (Sigma ChemicalCo.) with 0.1% RNase. The cells were once again incubated at roomtemperature for 30 minutes and then transferred to 4° C. and analyzedafter 18 hours by flow cytometry (Pallavicini, 1987). The resultsdemonstrate that prior to treatment of the cells withUA-BRF-004-DELEP-F035, 48% of the cells were in G0/G1 phase, 36% of thecells were in S phase and 7% of the cells were in G2/M phase. However,48 h post treatment of OVCAR-3 cells with F035, - 58% of the cells werein G1 and only 27% in S phase of the cell cycle, indicating an 8%increase of cells in G1 and ˜10% decrease of cells in S phase of thecell cycle (FIG. 19A, B). The results demonstrate a definite G1 arrestof OVCAR-3 human ovarian carcinoma cells

The effect of F035 on the cell cycle profile of human breast cancercells was also examined. MDA-MB-435 and MDA-MB-453 breast cancer cellswere exposed to different concentrations of F035 and analyzed 72 h laterby cell cycle analysis as described above. The results demonstrate thatF035 is inducing apoptosis of MDA-MB-435 cells by appearance of a Sub G0peak (Table 27). In addition cell cycle regulation is also observed byreduction in the percent of cells in S and G2/M phase of cell cycle.

TABLE 27 Cell Cycle Analysis of MDA-MB-435 Breast Cancer CellsPost-Treatment with F035 Control F035 (6 μg/ml) F035 (3 μg/ml) F035 (1μg/ml) Sub G0 0.82% 16.0% 12.7% 0.90% G1 52.0% 50.0% 50.3% 51.0% S 35.0%26.0% 26.0% 36.0% G2/M 16.0% 10.0%  2.0% 14.0%

Using the MDA-MB-453 cells, results demonstrate that F035 is inducing G1cell cycle arrest by a ^(˜)10% increase of cells in G1 phase and 4-10%decrease of cell in S phase of cell cycle seventy-two hours posttreatment with F035 (Table 28). These results demonstrate cell cyclearrest and apoptosis of tumor cells induced by the plant extract.

TABLE 28 Cell Cycle Analysis of MDA-MB-453 Breast Cancer Cells ControlF035 (6 μg/ml) F035 (3 μg/ml) F035 (1 μg/ml) Sub G0 0.96% 2.2%  1.8% 1.5% G1 62.0% 72.0%  71.0% 69.0% S 26.0% 19.0%  16.3% 21.0% G2/M 12.5%8.5% 10.4% 10.0%

Jurkat cells (1×10⁶) were treated with various concentrations ofUA-BRF-004-DELEP-F035 (50-1000 ng/ml) for 18 hours at 37° C. The cellswere washed once with PBS, resuspended in binding buffer (10 mMHepes/NaOH, 140 mM NaCl, 2 mM CaCl₂) containing 5 μl of annexin-V-FITCconjugate (Biowhittaker, Walkersville, Md.) and incubated for 10 minutesin the dark. The cells were washed and resuspended in binding buffercontaining 10 μl of 20 μg/ml propidium iodide (Sigma Chemical Co.) andanalyzed by florescence activated cell sorter (FACS) analysis (Martin etal., 1995).

Results demonstrate that the purified active compounds were able tocause apoptosis in Jurkat cells. This finding was further confirmed bythe ability of treated cells to bind annexin-V, an indication that cellswere undergoing apoptosis (Table 29). Normally, phosphotidylserine (PS)is localized on the inner membrane of the plasma membrane. However,during the early stages of apoptosis, externalization of PS takes place.Annexin-V is a calcium binding protein which binds to PS and can beobserved with annexin-V-FITC staining by flow cytometry (Martin et al.,1995).

TABLE 29 Apoptosis Measured by Annexin-V Binding in Jurkat Cells Treatedwith Various Concentrations of UA-BRF-004-DELEP-F035UA-BRF-004-DELEP-F035 (μg/ml) % Annexin-V positive cells Untreated 6Anti-Fas (positive control) 20.0 1 μg/ml 16.0 2 μg/ml 18.0

Example 11 UA-BRF-004-DELEP-F035 as a Chemoprotective Agent

The effect of UA-BRF-004-DELEP-F035 has been examined in a multi-stageskin carcinogenesis model in SENCAR mice. The animals were treated bypainting the skin with acetone, the carcinogen DMBA(7,12-dimethylbenz[a]anthracene), DMBA+UA-BRF-004-DELEP-F035, andDMBA+Fraction 60 (negative control) at low (100 μg ofUA-BRF-004-DELEP-F035 or Fraction 60 per mouse) and high (500 μg ofUA-BRF-004-DELEP-F035 or Fraction 60 per mouse) doses of plant extractadministered twice a week for 4 weeks. UA-BRF-004-DELEP-F035 or thecontrol was applied to the skin of mice 5 minutes before applying DMBA.The animals were observed for the formation of papillomas, and weresubsequently sacrificed and the tissues analyzed by histology (FIG.9A-FIG. 9F). The results of the analysis are summarized in FIG. 10A,FIG. 10B, FIG. 11A, FIG. 11B, FIG. 12 and FIG. 13.

After 8 weeks of these experiments, the group of mice treated with DMBAhad 8 papillomas per mouse, while those treated with DMBA andUA-BRF-004-DELEP-F035 had 0.66 papillomas per mouse, and those treatedwith DMBA and Fraction 60 (negative control) had 6.9 papillomas permouse. These results indicated a significant protective effect ofUA-BRF-004-DELEP-F035 against tumors, while there was essentially noprotective effect of Fraction 60.

Further murine in vivo studies demonstrated that UA-BRF-004-DELEP-F035is chemopreventative against carcinogen-induced tumors by preventing themutation of the ras oncogene. The initiation stage of carcinogenesis inmouse skin is accomplished by direct-acting carcinogens (i.e. DMBA) andis essentially an irreversible stage. Inhibition of carcinogenesis wasdetermined after 8 weeks by the reduction in formation of papillomasinduced by DMBA. Molecular analysis of the treated skin demonstratedthat UA-BRF-004-DELEP-F035 prevents DMBA's ability to mutate the rasoncogene (see Examples 14, 15 and 16, below).

Example 12 Procedure for Detection of Active Triterpenoids in Acaciavictoriae

A procedure was utilized which allowed the efficient detection of activetriterpenes in plant tissue sample. The procedure was carried out asfollows. Approximately 5 g of leaves and twigs were cut into smallpieces with scissors, or alternately, root samples were cut with a knifeto produce small slices. The plant material was processed in a smallblender, combined with approximately 25 ml of 80% methanol (v/v) andallowed to sit for at least 2 hours with shaking every 1 hour. Insolublematerial was removed by centrifugation at 10,000 g. The extract was thenused for thin layer chromatography with RP plates (aluminum TLC sheets,RP-C18 F_(254S)) and 40% acetonitrile (v/v). After exposure of the TLCplates to a 0.1% vanillin (4-hydroxy-3-methoxybenzaldehyde)/H₂SO₄ sprayand baking at 70° C. for 15 to 30 minutes, active triterpenoid compoundswere visible as brownish-red spots (R_(f)=0.2-0.3).

Example 13 Localization of Triterpene Compounds Within Acacia victoriaePlants

In initial studies, above-ground dry parts of the plants were collectedin early summer for extractions. Subsequent re-collection in the fallwas without activity. A systematic study was thereafter conducted todetermine for the relative absence or presence of the active triterpenecompounds in various parts of Acacia victoriae plants. After monitoringthe chemistry of the plant, it was determined that essentially all ofthe active components in the above-ground part of the plant wereconcentrated in the pods, roots and seedlings while largely orcompletely absent in the branches, bark, leaves and seeds. Therefore,the active collecting period only lasts about three weeks from the startof pod formation until dehiscence. It was also determined that the rootsof the plant produce the same active material with fluctuating ratios ofsugars to active components. The latter characteristic indicates thataeroponics, which allows for vigorous root growth while maintainingnormal plant development, may be well-suited for Acacia victoriae.

Example 14 Tumor Cell Lines and Growth Thereof

The following human cancer cell lines were obtained from American TypeTissue Culture Collection (ATCC, Rockville, Md.). SK-OV-3 and OVCAR-3(ovarian), Jurkat (T-cell leukemia), U-937 (histiocytic lymphoma),MDA-MB-468, MDA-MB-453, MDA-MB-435, SK-BP-3, MCF-7, MDA-MB-231, BT-20(breast), LNCaP, PC-3, DU145 (prostate), 769-P, 786-O, A498 (renal) andPANC-1 (pancreatic). HEY and Dov-13 (ovarian), cell lines were obtainedfrom M. D. Anderson Cancer Center. The following non-transformed humancell lines MCF-10A and 10F (breast epithelium) were obtained from M. D.Anderson Cancer Center. Hs 27 (human foreskin fibroblasts) and L929(mouse fibroblasts) were obtained from ATCC. SK-OV-3, MDA-MB-468, Hs 27,L929 were grown in minimal essential medium. OVCAR-3, Jurkat, U-937,LNCaP, DU-145, PC-3, HEY, Dov-13, PANG-1, MCF-10A, MCF-10F and remainingbreast cancer cell lines were grown in RPMI 1640 and F-12 media was usedto grow 769-P, 786-0 and A498. All the media used were supplemented with10% fetal calf serum, 200 mM glutamine and 0.05% gentamicin.

Example 15 Amplification of Mouse Ha-ras Codon 61 CAA→CTA MutationsUsing Mutation Specific Primers (MSP)

This protocol was derived from Nelson et al. (1992). A reverse primer,designated 3MSP61mut, was designed so that the 3′ end nucleotide (A)base pairs with the middle nucleotide (underlined) of a CAA→CTAtransversion in codon 61 of Ha-ras and selectively amplifies mutated DNAunder the conditions described below. The assay is based on the factthat Taq polymerase lacks 3′ exonuclease activity and thus cannot repaira mismatch at the 3′ end of the annealed primer. The conditions of theassay depend on the reverse primer failing to anneal sufficiently to thewild type sequence so that extension does not occur. Using the sameforward primer, one reaction is run with the reverse mismatch primer(3MSP61mut) and another reaction run with a reverse wild type primer(3MP61wt). This protocol detects only CAA→CTA transversions; howeverthese mutations are the most prevalent in codon 61 point mutations. AnXba I RFLP site (T/CTAGA) is created in this transversion. The mutationscan be verified by restriction of amplified DNA with Xba I or direct DNAsequencing using the ³²P end labeled 5MSP61 primer. The reactionscontaining the mismatch products can be run on 2% low melt agarose forsubsequent purification and sequencing. The sequence of the primersused, 5MSP61, 3MSP61mut, and 3MSP61wt, is given below and in SEQ IDN0:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

5MSP61 (23-mer) 5′-CTA AGC CTG TTG TTT TGC AGG AC-3′ (SEQ ID NO: 2)3MSP61mut (20-mer) 5′-CAT GGC ACT ATA CTC TTC TA-3′ (SEQ ID NO: 3)3MSP61wt (20-mer) 5′-CAT GGC ACT ATA CTC TTC TT-3′ (SEQ ID NO: 4)

The sequence of 3MSP61wt has 2 or 3 mismatches from N-ras and K-rassequences, respectively, fragment size is 110 bp. The template DNA andamplification reagents were as follows:

DNA (Positive Control) OR 1.0 μg DNA (Negative Control, i.e. wild type)OR 1.0 μg DNA (Sample i.e. paraffin block) OR 5.0 μl No DNA (i.e. H₂O)5.0 μl Rxn Buffer (10X) (10X = 500 mM KCl, 5.0 μl 100 mM Tris, pH 8.3,15 mM MgCl₂) dNTP mixture @ 500 μM each 2.0 μl (final conc. = 20 μM)[³²P] dCTP, 3000 Ci/mmol, 0.50 μl 5 uCi, 1.7 pmol, 0.034 μM 5′ Primer(10 pmol/μl), 7.5 pmol 0.75 μl (final conc. 0.15 μM) 3′ Primer (10pmol/μl), 7.5 pmol 0.75 μl (final conc. 0.15 μM) Taq Polymerase (5 U/μl,3.0 U) 0.60 μl H₂O to 50.0 μl 50.0 μl Mineral Oil 2 drops

The amplification cycle conditions, using a Perkin Elmer thermocycler,were as follows:

Preheat thermocycler to 95° C. File 512-21 95° C. 1 min 30 sec 1 CycleFile 512-22 95° C. 60 sec 57° C. 60 sec 72° C. 60 sec 30 Cycles File512-10 Soak 4° C.

Validation of the assay was accomplished by running the followingcontrols: Wild Type (WT), Wild Type Mutant (MUT), and negative control(H₂O). MUT DNA from the plasmid pHras61mut was used as a positivecontrol sample. The plasmid pHras61 contains cloned exon 2 Ha-ras DNAfrom a Senear mouse tumor. The cloned mutation was verified by DNAsequencing. The mutation is the CA→CTA transversion in codon 61 (locatedin exon 2) of the mouse Ha-ras gene is a sample of DNA from tumoradenocarcinoma containing Ha-ras mutation in codon 61 (See FIG. 14).

Example 16 Hot PCR™/RFLP Mutation Assay for Mouse H-ras Codon 12/13

This assay is based on disruption of a naturally occurring Mnl I sitespanning the three by of codon 12 and the first by of codon 13 (GGA GGC,nucleotides 34-37 in the rat and mouse Ha-ras coding sequence). Therecognition site for Mnl I is N7GGAG. Mutations in any of these fourpositions will result in failure of Mnl I to cut the PCR™ fragmentcontaining this region. The drawback of the assay is the occurrence ofincomplete digestion by Mn1 I. Such an event makes it difficult todistinguish between a small percentage of wild type DNA resistant todigestion and a low level of genuine mutations. This is sometimesobserved when the source DNA contains a mixture of wild type and mutantDNA and when the assay employs ³²P for fragment labeling to increasesensitivity. The PCR™ Primer Set used for the is assay is given below,and in SEQ ID NO:5 and SEQ ID NO:6. The H-ras 12 amplification productsize is 214 bp.

Primer #3 (5′): 5′-CCTTGGCTAAGTGTGCTTCTCATTGG-3′ (SEQ ID NO: 5) Primer#6 (3′): 5′-ACAGCCCACCTCTGGCAGGTAGG-3′ (SEQ ID NO: 6)

Primer #6 is used for sequencing at 55° C. using the following reactionconditions:

Rxn Buffer (10X) 1.0 μl (10X = 500 mM KCl, 100 mM Tris, pH 8.3, 15 mMMgCl2 dNTP mixture @ 0.5 mM each 1.0 μl 5′ Primer 6 pmol 3′ Primer 6pmol ³²P-dCTP (3000 Ci/mMol) 0.5 μl Taq Polymerase (5 U/μl, 0.65 U) 0.13μl H₂O to 10.0 μl DNA (Positive Control) >200.0 ng DNA (Negative Controli.e. wild type) >200.0 ng DNA (Sample i.e. paraffin block) 5.0 μl No DNA(i.e. H₂O) 5.0 μl Mineral Oil 2 drops

The amplification cycle conditions, using a Perkin Elmer PCR™ Kit, areas follows:

Preheat thermocycler to 94° C. File 512-87 94° C. 2 min 1 Cycle File512-88 94° C. 30 sec 68° C. 30 sec 72° C. 1 min 8 Cycles File 512-89 94°C. 30 sec 60° C. 30 sec 72° C. 1 min 32 Cycles File 512-10 Soak 4° C.

Example 17 Assay Results: Detection of c-Ha-ras Mutations

Four days after the last administration of DMBA, the plant extract andthe control, DNA isolated from freshly-frozen tissues of 5 mice pergroup was analyzed for mutations in codons 12 and 13, and codon 61 ofc-Ha-ras by PCR™ analysis. The inventors have used 4-day specimens forthis analysis-because some of the 2-day DNAs were degraded and thereforenot suitable for Ha-ras analysis. In codons 12 and 13 there is a Mnl Irestriction site which spans the three nucleotides of codon 12 and thefirst nucleotide of codon 13 in the wild type sequence. Mutations in anyof these bases result in the loss of the Mnl I site. The inventorsamplified exon 1 (which contains codons 12 and 13) of the c-Ha-ras genefrom genomic DNA using a Perkin-Elmer thermal cycler. The reaction wasextracted with phenol-CHCl₃ and the DNA was precipitated with ethanol.The DNA was then resuspended in enzyme buffer, and the PCR™ product wasrestricted with Mn1 I and the digest electrophoresed on a 8%nondenaturing polyacrylamide gel. No loss of Mn1 I restriction site wasobserved and the conclusion was that there are no mutations in codons 12and 13 in the tested material. DNA for Ha-ras analysis was also obtainedfrom paraffin-embedded sections cut a 8μ from samples collected two daysafter last dosing. The 25 sections from each paraffin block were placedin microfuge tubes, deparaffinized with xylene and ethanol, centrifugedand resuspended in 5% chelex with proteinase K.

The first procedure used for Ha-ras codon 61 was derived from Nelson etal. 1992 (Example 15, above). Using the same forward primer, onereaction was run with the reverse mismatch primer (3MSP61mut) andanother reaction was run with a reverse wild type primer (3MSP61wt).This protocol detects only CAA→CTA transversion mutations that are themost prevalent in codon 61 point mutations. An Xba I RFLP site (T/CTAGA)is created in this transversion. The reactions containing the mismatchproducts were run on 2% low melt agarose for subsequent purification andsequencing. The ratio of the amount of cut (wild type DNA) to uncut(mutated DNA) was determined by quantifying ethidium bromide stainingintensity or ³²P labeling. The DNA from the plasmid pHras61mut was usedas a positive control sample. The plasmid pHras61 contains cloned exon 2Ha-ras DNA from a Sencar mouse tumor. The cloned mutation was verifiedby DNA sequencing. The mutation is the CAA→CTA transversion in codon 61(located in exon 2) of the mouse Ha-ras gene. The reaction conditionswere as described in Example 15.

Example 18 Effect of F035 on the Initiation of Aberrant Crypts in F344Rats Treated with Azoxymethane

Male rats (Fishcer, 3444) were obtained from Charles River (Raleigh,N.C.) at 6 weeks of age. The rats were fed ad libitum an AIN-76A dietthat was purchased from Dyets Inc. (Bethlehem, Pa.). The diet consistedof 20% casein, 0.3% DL-methionine, 15% corn starch, 50% sucrose, 5% cornoil, 5% cellulose, 3.5% AIN-76 salt mix, 1% AIN-76 vitamin mixture and0.2% choline bitartrate. The animals were also provided with tap waterad libitum. Azoxymethane (AOM), which induces aberrant crypts in rats,was purchased from Sigma Chemical Company (St. Louis, Mo.). Animals werefed rat chow for three days while in quarantine and then they were fedAIN-76A until 7 wk of age. The animals were randomized into threetreatment groups (10 animals/group). The animals in group 1 were fedwith AIN-76A diet alone, group 2 animals received AIN-76A diet+5 mg/kgof F035 and the animals in group 3 were fed the AIN-76A diet+10 mg/kg ofF035 (Table 30).

TABLE 30 Treatment Groups for Study on the Effect of F035 on theInitiation of Aberrant Crypts in F344 Rats Using Azoxymethane Group #Animals F035 Dose (mg/kg diet) 1 10 0 2 10 5 3 10 10

One week following feeding all the animals were given intraperitonealinjection of AOM (15 mg/kg body weight). The second AOM injectionfollowed one week later. Animals were weighed weekly throughout thestudy. The animals were fed for 4 weeks. Thirty-one days later theanimals were sacrificed by CO₂ asphyxiation. The colons were excised andflushed with cold PBS, cut along the longitudinal median axis, placed ona filter paper and fixed in 70% alcohol for at least 24 h. The colonswere stained with methylene blue (0.25% in PBS)for ˜1 min. Aberrantcrypt foci were scored under a dissecting microscope at 20×. Theaberrant crypts were distinguished from the surrounding normal crypts bytheir increased size, significantly increased distance from the luminalto basal surfaces of cells and enlarged pericryptal zone. All thespecimens were coded and scored blindly by two scorers. Statisticalsignificance was determined by checking for the differences between thegroups using one way ANOVA. If the differences were found, a bonferronit-test was used to test multiple comparisons of both doses of F035 tothe control group. The scoring of the colons was done by two scorers,each blinded as to the experimental groups they were scoring. There wasgood agreement between the two scorer's results.

It was found that F035 significantly reduced the total number ofaberrant crypt foci when added to the diet at 10 mg of F035 per kg ofdiet, which is roughly equivalent to a daily intake of one mg of F035per kg of body weight (Table 31, Table 33). The same dose alsosignificantly reduced the number of aberrant crypts in the singlets anddoublets categories (Table 32, Table 34). The lower dose of F035, 5 mgper kg of diet (roughly equivalent to a daily intake of 500 microgramsof F035 per kg of body weight) did cause a reduction in the total,singlets and doublets categories of aberrant crypt foci, but thereduction was not significantly different from the control values (Table32, Table 34). There was no difference in weight gain between theexperimental and control groups over the course of the study (Table 35,Table 36, Table 37).

TABLE 31 Effect of F035 on Number of Aberrant Crypts/Colon inAOM-Treated Rats Aberrant Crypts/ Dose of F035 Colon Averaged Scorer 1 &2 g/kg diet Means ± SEM % Control Result Comments 0 86 ± 5 100 0.005 73± 5 85 − C 0.01 43 ± 4 50 + C − = Not significantly different fromcontrol + = Significantly different from control (p < 0.05) C =Conclusive study

TABLE 32 Effect of F035 on Number of Aberrant Crypts per Focus in AOM-Treated Rats Number of Aberrant Crypts Per Focus (Averaged Scorer 1 and2) Agent & (g/kg 1 2 ≦3 Dose Diet) Mean ± SEM % Mean ± SEM % Mean ± SEM% Fraction 35 0 66 ± 5 100 17 ± 1 100 3 ± 1 100 Fraction 35 0.005 56 ± 485 15 ± 1 88 3 ± 0 100 Fraction 35 0.01  34 ± 3* 52  8 ± 1* 47 1 ± 0 33*significantly different from control (p < 0.05)

TABLE 33 Summary of Raw Data from Analysis of the Effect of F035 on MeanAberrant Crypts Per Colon in AOM Treated Rats Dose Mean Aberrant g/kgColon ± SEM Crypts/ n AOM Agent diet Scorer 1 Scorer 2 Combined 10 +Carcinogen 0 76 ± 4 95 ± 9 86 ± 5 only (4 weeks)^(a) 10 + F035 0.005 67± 4 80 ± 8 73 ± 5 10 + F035 0.01  34 ± 3^(c)  51 ± 7^(c)  43 ± 4^(c)^(a)AOM injected rats; no test agent ^(b)Average of pooled AOM injectedrats (n = 10) at 4 weeks ^(c)significantly different from control (p <0.05)

TABLE 34 Summary of Raw Data from Analysis of the Effect of F035 onNumber of Aberrant Crypts Per Focus in AOM Treated Rats Number ofAberrant Crypts Per Focus Mean ± SEM Agent Dose Scorer 1 2 3 Total F035 5 scorer 1 49 ± 4 15 ± 2 2 ± 0 67 ± 4 mg scorer 2 63 ± 7 15 ± 1 3 ± 180 ± 8 combined 56 ± 4 15 ± 1 3 ± 0 73 ± 5 10 scorer 1  27 ± 3*  7 ± 1* 0 ± 0*  34 ± 3* mg scorer 2  41 ± 5*  8 ± 1* 3 ± 1  51 ± 7* combined 34 ± 3*  8 ± 1* 1 ± 0  43 ± 4* Control NA scorer 1 57 ± 4 17 ± 1 3 ± 176 ± 4 scorer 2 75 ± 8 18 ± 2 3 ± 1 95 ± 9 combined 66 ± 5 17 ± 1 3 ± 186 ± 5 *Significantly different from control values (p < 0.05)

TABLE 35 Animal Weights of AOM-Treated Rats Fed F035, 5 mg/kg diet Rat #Week 1 Week 2 Week 3 Week 4 Week 5 1 154.4 198.2 215.0 219.7 235.8 2149.8 195.1 210.6 210.6 232.9 3 154.1 200.7 228.1 228.1 248.0 4 154.1199.8 216.2 220.8 242.9 5 158.0 208.4 228.4 231.5 256.8 6 154.8 196.0208.3 213.4 230.2 7 164.2 210.1 224.4 225.5 246.8 8 161.7 202.3 218.8220.4 237.8 9 153.0 199.7 217.0 218.1 238.1 10  158.8 198.5 212.8 212.3231.6 Mean 156.3 200.9 218.0 220.0 240.1 SEM 1.4 1.6 2.2 2.2 2.7

TABLE 36 Animal Weights of AOM-Treated Rats Fed F035, 10 mg/kg diet Rat# Week 1 Week 2 Week 3 Week 4 Week 5 1 148.6 187.1 201.9 205.6 224.8 2148.3 189.9 196.0 199.0 220.8 3 149.0 197.7 211.2 216.2 237.2 4 146.2189.1 206.1 209.2 230.0 5 151.9 197.2 214.9 218.6 241.2 6 152.2 190.0205.2 208.1 226.6 7 136.1 187.8 211.8 216.2 241.2 8 157.4 207.1 224.1224.8 246.0 9 141.9 187.8 207.7 211.1 235.6 10  155.7 185.9 196.4 194.9213.9 mean 148.7 192.0 207.5 210.4 231.7 SEM 2.0 2.1 2.7 2.9 3.2

TABLE 37 Animal Weights of AOM-Treated Rats Rat # Week 1 Week 2 Week 3Week 4 Week 5 1 149.3 195.2 203.2 214.4 240.6 2 166.6 213.8 229.8 231.8250.7 3 158.6 195.8 209.0 211.2 226.8 4 156.4 200.3 214.3 216.9 231.2 5151.2 194.7 205.2 207.4 228.5 6 157.3 203.9 217.2 217.8 237.0 7 146.7192.1 216.6 217.1 235.2 8 145.5 190.3 203.8 204.7 220.3 9 158.1 197.4212.3 211.3 231.2 10  157.7 201.8 217.9 219.2 240.8 Mean 154.7 198.5212.9 215.2 234.2 SEM 2.0 2.2 2.6 2.4 2.7

Example 19 Antitumor Activity of Aglycones

Studies confirm the importance of the sugar to the biological activityas the removal of the sugars from the core triterpene molecule resultsin significant loss of biological activity. As shown in Table 38,UA-BRF-004Pod-DELEP-F164 (generated by hydrolysis of the sugars fromUA-BRF-004Pod-DELEP-F094 with esters attached) andUA-BRF-004Pod-DELEP-F245 (a methyl ester mixture of the hydrolysisproduct of UA-BRF-004Pod-DELEP-F094) show marked loss of anti-tumoractivity against a panel of tumor cell lines. Similarly,UA-BRF-004Pod-DELEP-C 194 (the purified acetate of aglycon 1) exhibitssubstantial of anti-tumor activity against a panel of tumor cell linescompared to the data of the triterpene glycoside Fraction 35. Thus,there is a marked loss of biological activity following hydrolysis ofthe sugar units from the triterpene glycoside disclosed herein.

TABLE 38 Bioassay of Fractions F164, F245 and C194. 50 μg/ml 25 μg/ml12.5 μg/ml 6.25 μg/ml 3.12 μg/ml F164 769-P 45 20 0 0 0 Panc-1 57 27 130 0 Dov-13 80 56 16 12 10 MDA-MB-453 66 30 13 0 0 JURKAT 93 86 55 3916.5 F245 769-P 26 14 14 7 0 Panc-1 49 26 4 0 0 Dov-13 91 90 25 28 13MDA-MB-453 90 75 8 8 0 JURKAT 93 89 64 23 0 C194 769-P 13 6 0 0 0 Panc-16 9 0 0 0 Dov-13 3 0 0 0 0 MDA-MB-453 16 0 0 0 0 JURKAT 34 18 9 2 0

Example 20 Analysis of the Effect of F035 on Cholesterol Metabolism

The purpose of this study is to analyze the effect of the biologicallyactive triterpene glycosides of the invention on the prevention ofcardiovascular disease. The long-term objective of this study is todemonstrate that the triterpene compounds added to the diets of mammals,including humans, will reduce serum cholesterol. The hyperlipidemichamster model selected for the study is a rodent model, which incontrast to the rat model, closely mimics both the LDL receptor andhuman plasma lipoprotein changes in response to cholesterol content(Spady et al., 1993).

The triterpene glycoside is administered, at two differentconcentrations, into a purified hamster diet without any change in thelevel of calcium, potassium, phosphorus or other essential components ofthe diet. Two different levels of supplementation with triterpeneglycoside are used in order to show a dose-response relationship. Theanimals are fed with free access to the Dyets purified hamster dietformulated according to NRC recommendations (Reeves et al., 1993) withor without 1% cholesterol (Davis et all, 1989). The Dyets purifiedhamster diet containing cholesterol is modified with triterpeneglycoside using concentrations indicated below. Pelleted study andcontrol diets are prepared by Dyets, Inc. (Bethlehem, Pa.) with nochange in the content of calcium, phosphorus or any essentialmicronutrient. Animals are monitored for food intake and body weightgain weekly. The animals used are four-week old, male outbred,virus-free Golden Syrian hamsters (Charles River Laboratories,Wilmington, Mass.). Animals are randomized by weight using a randomnumber generator in the Statview program, housed 3 per cage in a roomilluminated 12 h per day and maintained at a temperature of 22° C.±1.0°C.

After 0, 4, and 8 wk on their respective diets with or without thetriterpene glycoside, 12 animals per group are selected at random andkilled at 9-11 a.m. The liver and kidneys are removed, weighed,processed, and stored at −70° C. for future studies. Blood is obtainedat sacrifice by cardiac puncture prior to the removal of the liver andkidneys and analyzed for lipid profiles. The blood serum lipid profilesare analyzed between the treatment and control groups. There are twocontrol groups (Groups 1 and 2) and two treatment groups (Groups 3 and4). All the groups receive the NRC hamster diet during a two-weekquarantine period. Group 1 continues on the NRC diet until the end ofthe study. Groups 2-4 are fed the NRC diet plus 1% cholesterol foranother two-week period to induce hypercholesterolemia. Then, Group 2will continue on this diet until the end of the study, while Groups 3and 4 will be fed the same diet supplemented with the triterpeneglycoside (e.g., F035 or F094). A summary of the treatment groups isgiven below, in Table 39.

TABLE 39 Scheme of Diet Modification Initial Number of Diet ModifierGroup No. Hamsters Symbol Concentration 1 24 + 12^(a) None — 2 24 +12^(a) Chol^(b)+ 1% Chol 3 24 Chol^(b) + TG^(c) 1% Chol + 0.003% TG 4 24Chol^(b) + TG^(c) 1% Chol + 0.075% TG ^(a)To be sacrificed at thebeginning of triterpene glycoside feeding. ^(b)Chol = cholesterol ^(c)TG= triterpene glycoside

After 0 (control group only), 4, and 8 wk on their respective diets,with or without the triterpene glycoside, 12 animals per group areselected at random and killed at 9-11 a.m. The livers and kidneys ofhamsters were removed, weighed and processed for possible abnormalities.A portion of each organ showing abnormalities was prepared for histologyanalysis, i.e., frozen for paraffin sections and sections stained withhematoxylin and eosin. Blood was obtained at sacrifice by cardiacpuncture prior to surgical removal of the liver and kidneys. Serum wasprepared and kept at −20° C. for lipid profile analysis. Hamsters werefasted overnight prior to sacrifice. Data is shown in Table 40 below.

Blood samples collected in the course of this study are used fordetermination of total cholesterol, triglycerides, HDL-cholesterol, andLDL-cholesterol plus VLDL-cholesterol (Mackness and Durrington, 1992) atthe Roche Biomedical Laboratories, Burlington, N.C. Statistical analysisof the data is performed on a Power Macintosh 9600 computer withMacintosh software for one-way analysis of variance, p value, and linearregression (Armitage, 1971). In particular, data analysis of lipidprofiles in each diet/drug group is performed by analysis of varianceusing Newman-Keuls mean separations (Steel and Torrie, 1980).

TABLE 40 Effect of Continual Feeding of Triterpene Glycoside (TG) toHamsters for Six Weeks Total HDL LDL Cholesterol TriglyceridesCholesterol Cholesterol (mg/dL) (mg/dL) (mg/dL) (mg/dL) % % % % DietGroup Average Change Average Change Average Change Average ChangeControl 141 — 133 141 — 0 — Cholesterol 341 — 247 281 — 31 — 0.015% TG329 −3.5 260 5.3 250 −11 36 16.1 0.03% TG 303 −11.1 236 −4.4 246 −12.415 −51.6 12 hamsters/group fed purified hamster diet plus 1% cholesterol

Example 21 Study on the Prevention of UVB-Induced Carcinogenesis withFraction 35

This study will focus on the prevention of UVB-induced carcinogenesis inthe mouse skin model with the active triterpene glycosides of theinvention. The long-term objective of the study is to demonstrate thatin the mouse skin model the triterpene glycosides will preventUVB-induced lesions. The mouse experimental model is used because themodel closely resembles the human situation. In the study, the inventorswill seek to demonstrate that topical application of the activetriterpene compounds of the invention in acetone to the dorsal skin ofSKH-1 hairless mice irradiated with UVB will prevent skin lesions causedby UVB.

In the study, SKH-1 hairless mice are irradiated with UVB radiation atthe dose of 1.8 kJ/m² for up to 15 min. Mice are pretreated with twodifferent doses of F035 of (2 mg and 4 mg per dose) as well as negativecontrols (F060 or acetone alone). It is believed a minimum of 10 miceper group are needed to obtain statistically meaningful results. Eachtest compound is applied topically 10 min before irradiation 3 times perwk for up to 6-10 wk. The studies are conducted for a short period oftime to evaluate the preventive effect of the compounds. It is notexpected to see visible tumors, even with UVB alone, only skin lesionswithin the specified time-frame. A slight erythema (minor redness of theskin) may be observed, which should disappear the next day afterirradiation.

The UV apparatus used has eight Westinghouse FS40 sunlamps, an IL-1400Aradiometer/photometer, and an attached IL-1403 UVB phototherapyradiometer with a SEL 240/UVB-1/TD detector. The middle part has severalchambers, each holding an individual mouse. There are holes inside thechambers for proper ventilation while mice are being irradiated. Thechambers rotate in circular motion during irradiation so each mouse isexposed to UVB light uniformly. There are doors in this device thatcould be closed while the UVB lamp is on, so the UVB light is containedinside the device. The amount of the UVB exposure will be measured witha UVB radiometer. Mice should stay in the chambers for not longer than10 to 15 min.

The purpose of the study is to establish photoprotective effects againstUVB injury in mouse skin. UVB is absorbed directly by cellular DNA andproduces lesions that may cause mutations in the target gene(s),ultimately leading to cancer. Early detection of these lesions andprevention of such lesions may indicate chemoprotective effects (Bertonet aL, 1997; Chatterjee et al., 1996; Youn et al., 1997; Shirazi et al.,1996; Baba et al., 1996; Takema et al., 1996).

TABLE 41 UVB-Irradiation Regimen UVB alone 10 mice per group Acetone/UVB10 mice per group F035 (2 mg/dose) 5 to 10 min later UVB 10 mice pergroup F035 (4 mg/mouse) 5 to 10 min later UVB 10 mice per group F060 (2mg/mouse) 5 to 10 min UVB 10 mice per group F060 (4 mg/mouse) 5 to 10min UVB 10 mice per group

The treatment groups for the study are as indicated in Table 41. Thesize of the groups is deemed sufficient to control variation in skinhyperplasia and skin inflammation in a given group, inter-animalvariation in epidermal thickness and skin inflammation in animals of thesame age and the same developmental stage. Hyperplasia and skininflammation are the main parameters measured in the study. Remainingskins are preserved for measurements of other biomarkers, like modifiedDNA bases (8-OH-dG) and oncogene expression (Ha-ras oncogene).

The animals will have free access to pelleted diets and drinking waterthroughout the study. Animals will be monitored for food intake and bodyweight gain weekly. The animals used are seven-week old, female outbred,virus-free SKH-1 hairless mice (Charles River Laboratories, Wilmington,Mass.). Animals are randomized by weight using a random generator in theStatview program, housed 5 per cage in a room illuminated 12 h per dayand maintained at a temperature of 22° C.±1.0° C.

Statistical analysis of data is performed on a Power Macintosh G3computer with Macintosh ‘software for one-way analysis of variance, pvalue, and linear regression (Armitage, 1971). In particular, dataanalysis of epidermal thickness in each drug group is performed byanalysis of variance (Armitage, 1971).

Example 22 Effect of Biologically Active Triterpenes on the Expressionof Proteins Involved in

Cell-Cycle Arrest and Apoptosis

Apoptosis is defined as a normal physiologic process of programmed celldeath which occurs during embryonic development and during maintenanceof tissue homeostasis. The process of apoptosis can be subdivided into aseries of metabolic changes in apoptotic cells. Individual enzymaticsteps of several regulatory or signal transduction pathways can beassayed to demonstrate that apoptosis is occurring in a cell or cellpopulation, or that the process of cell death is disrupted in cancercells. The apoptotic program is also observed by morphological featureswhich include changes in the plasma membrane (such as loss ofasymmetry), a condensation of the cytoplasm and nucleus, andinternucleosomal cleavage of DNA. This is culminated in cell death asthe cell degenerates into “apoptotic bodies”.

Techniques to assay several enzymatic and signaling processes involvedin apoptosis have been developed as standard protocols formultiparameter apoptosis research. One example of an early step inapoptosis, is the release of cytochrome c from mitochondria followed bythe activation of the caspase-3 pathway (PharMingen, SanDiego, Calif.).Induction of the caspases (a series of cytosolic proteases) is one ofthe most consistently observed features of apoptosis. In particular,caspase-3 plays a central role in the process. When caspases areactivated, they cleave target proteins; one of the most important ofthese is PARP (a protein located in the nucleus). Therefore, assaysdetecting release of cytochrome c, detecting caspase-3 activity anddetecting PARP degradation are effective determinants of apoptosis.

Furthermore, agents that cause the release of cytochrome c from themitochondria of malignant cells can be concluded to be likely therapiesfor restoring at least some aspects of cellular control of programmedcell death.

Another apoptotic assay is the Annexin-V detection (BioWhitaker,Walkerville, Md.). Normally, phosphotidylserine (PS) is localized on theinner membrane of the plasma membrane. However, during the early stagesof apoptosis, externalization of PS takes place. Annexin-V is a calciumbinding protein which binds to PS and can be observed withannexin-V-FITC staining by flow cytometry (Martin et al., 1995). Theability of cells treated with the Acacia victoriae compounds describedin this invention, to bind annexin-V, is taken as an indication thatcells were undergoing apoptosis.

In other examples, the inventors have used PI-3-Kinase assay and todetect the apoptotic activity in cells treated with the anti-cancercompounds isolated from Acacia victoriae. Phosphoinositide 3-kinase(PI3K), a cell membrane associated enzyme, is capable of phosphorylatingthe 3-position of the inositol ring of phosphatidylinositol, thusdefining a new lipid signaling pathway in those cells where PI3K isactive. When PI3K is active, a kinase called AKT is recruited to thecell membrane. AKT is the product of an oncogene which is catalyticallyactivated after recruitment to the membrane. Fully activated AKT plays acrucial role in cell survival. The PI3K/AKT pathway provides a mechanismby which cells evade apoptosis. Thus, a means to inhibit PI3K inmalignant cells, is a likely therapy for restoring at least some aspectsof the cellular control of apoptosis.

Example 23 Cell Cycle Analysis

Cell cycle analysis was done by flow cytometry by standard methods withsome modifications. Briefly, 1×10⁶ cells were plated in 60-mm³ dishesand exposed to various concentrations of F035 for 72 h at 37° C. Cellswere washed in PBS and resuspended at a concentration of 1×106 cells/ml.Cells were fixed first with 1% paraformaldehyde followed by ice cold 70%ethanol. The cells were then stained with propidium iodide (10 μg/ml;Sigma Chemical Co., St. Louis, Mo.) containing 0.1% RNAse (Sigma) for 30min at room temperature and analyzed on a Beckton Dickinson FAC SCAN.

Example 24 AnnexinV- Fluorescein Isothiocyanate (FITC) Binding Assay

Induction of apoptosis in cancer cells was studied by AnnexinV- FITCbinding assay. Jurkat cells (1×10⁶) were treated with variousconcentrations of mixture of triterpene glycosides (F035) and pureextracts D1 and G1 (0.5-2.0 μg/ml) for 18 h at 37° C. After washing thecells in PBS they were resuspended in binding buffer (10 mM HEPES/INaOH,140 mM NaCl, 2 mM CaCl₂) containing 5 μl of annexin V-FITC conjugate(Bio Whittaker, Walkersville, Md.) and incubated for 10 min in the dark.Cells were next stained with propidium iodide (20 μg/ml) and analyzed byflow cytometry. (Martin et aL, 1995).

Example 25 Phosphatidylinositol 3-Kinase (PI3-Kinase) Assay

The serum starved Jurkat cells were treated with 2 of F035 for 2 -15 hor 0.5 h with wortmannin at 37° C. PI 3-kinase activity was determinedas described (Whitman et al., 1985; Royal and Park, 1995). PI3-kinasewas immunoprecipitated from 1 mg of cellular protein using 5 μl rabbitanti p85 antiserum at 4° C. for 90 min. The immune complexes werecollected on 20% protein A-sepharose beads for 90 min at 4° C. Next theimmunoprecipitates were resuspended in 30 μl of kinase reaction buffer(33 mM Tris, pH 7.6, 125 mM NaCl, 15 mM MgCl₂, 200 mM of adenosine, 20mM ATP, 30 uCi [g-32] adenosine triphosphate ATP). The PI3-kinasereaction was initiated by addition of 10 μl of the PI suspension and 10μl of gamma-ATP and allowed to proceed for 30 min at room temp. Adding100 μl of 1 N HCl terminated the reaction. Lipids were extracted fromthe reaction mixture with chloroform: methanol (1:1) and resolved bythin layer chromatography (TLC) in chloroform: methanol: NH₄OH: H₂O(60:47:2:11.3) on silica gel G60 plates. Radio labeledphosphatidylinositol (PI) phosphate was visualized by autoradiographyand inhibition was quantitated using a Storm 860 system (MolecularDynamics).

Example 26 Analysis of the Total and Phosphorylated forms of AKT

The expression of total and phosphorylated forms of AKT was determinedby western blot analysis. Jurkat cells cultured in medium containing0.5% FBS were treated with F035 and pure extracts DI and G1 (2.0 μg/ml)for 15 h at 37° C. The cells were lysed in AKT Iysis buffer (20 mMTris-HCl, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mMsodium pyrophosphate, 1mM 8-glycerol phosphate, 1 mM Na₃VO₄, 1 mlleupeptin, 1 mM PMSF pH 7.5). Cellular protein (40 μg) was resolved on8% SDS-polyacrylamide gel and electrotransferred onto nitrocellulosemembrane. Membranes were probed first with phosphospecific AKT (Ser 473)or AKT antibody followed by goat anti rabbit antibody conjugated tohorseradish peroxidase. Proteins were detected by chemiluminescence(ECL, Amersham, Arlington Heights, Ill.).

Example 27 Electrophoretic Mobility Shift Assay (EMSA)

An EMSA to study the effect of crude (F035) and pure extracts D1 and G1on TNF (Genetech Inc.) induced NF-κB was done as described earlier.Jurkat cells (1×10⁶/ml) were treated with different concentrations ofcrude and pure extracts for 15 hr at 37° C. Next the cells were exposedto 100 pM of TNF for 15 min at 37° C. Nuclear extracts were prepared asdescribed before. Nuclear extracts were incubated with 16 fmol³²P-end-labeled 45-mer double-stranded NF- κB oligonucleotide from thehuman immunodeficiency virus long terminal repeat,

(SEQ. ID NO. 9) 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCTGG-3′

for 15 min at 37° C. in the presence of 2 μg of poly (dI-dC). The DNAprotein complex was separated from free oligonucleotide on 7.5% nativepolyacrylamide gels. The radioactive bands from the dried gels werevisualized and quantitated by a Phospholmager (Molecular Dynamics,Sunnyvale, Calif.) using ImageQuant software.

Example 28 Induction and Analysis of Inducible Nitric Oxide Synthase(iNOS)

U-937 and Jurkat cells were used for studying iNOS. U-937 cells weredifferentiated into macrophages by culturing them with PMA (100 nM) for72 hr at 37° C. The differentiated cells were treated with F035 (2μg/ml) for 15 hr followed by a 4 hr treatment with LPS (10 μg/ml) induceiNOS. In Jurkat cells the iNOS was induced by treating 0.5×10⁶/ml cellswith PHA (10 μg/ml) and PMA (10 ml) for 24 hr at 37° C. Cell lysateswere prepared by repeated freezing and thawing in RIPA buffer (1% NP-40,0.5% Na deoxycholate, 0.1% SDS in PBS). Cellular protein (200 μg) wasresolved on a 7.5% SDS-polyacrylarnide gel, electrotransferred ontonitrocellulose membrane, probed with rabbit anti-iNOS antibody followedby goat anti-rabbit antibody conjugated to horseradish peroxidase.Protein bands were detected by chemiluminescence (ECL, Amersham,Arlington Heights, Ill.).

Example 29 Mixture and Pure Triterpene Glycosides Induce Tumor CellCytotoxicity

The effect of the mixture of triterpene glycosides (F035) on theviability of a panel of cancer and non-transformed cells was studied asdescribed in the methods. As shown in FIG. 42, Jurkat (T-cell leukemia)cells were highly sensitive to F035 with an IC₅₀ of 0.2 μg/ml. SimilarlyF035 inhibited the growth of number of cancer cell lines with inhibitoryconcentration IC₅₀ in range of 1.7-2.8 for (ovarian), 2.0-3.3 (renal),0.93 (pancreatic), 1.2-6.5 (prostate) and 0.72-4.0 ml (some breast)cancer cells. However remaining breast cancer cell lines were resistantto cytotoxic effect of F035. The last four bars of FIG. 42 show thatmore than 25 g/ml of F035 was required to kill 50% of non-transformed(human and mouse fibroblasts and immortalized breast epithelium) cellssuggesting that F035 is specifically cytotoxic the cancer cells.

In addition, two pure triterpene glycosides DI & G1 were tested forcytotoxicity on 5 cell lines. FIG. 43 shows that D1 has an IC₅₀ that iscomparable to F035 in three cell lines (769-P, MDA-MB- 453, &MDA-MB-231). In C-2 (HEY Variant) and Jurkat cells, D1 is twice aspotent as F035. However, G1 was significantly more cytotoxic than F035and D1 in most of the cells tested which could be because G1 is lesspolar than extract D1 (FIG. 43).

Example 30 Cell Cycle Arrest and Induction of Apoptosis with Mixture ofTriterpene Glycoside

To study the effect of F035 on cell cycle, cancer cell lines MDA-MB-453and MDA-MB-435 were treated with different concentrations F035 . Table42 showed an increase in the number of cells in G1 (7-10%) and aconcomitant decrease in the %. of cells in S phase (6-10%) suggesting aGI arrest in MDA-MD-453 cells. In addition, after 72 hr post treatmentwith F035, 16% of MDA-MB435 (another breast cancer cell line) cellsappeared to be in SubG_(o) phase of cell cycle (Table 42) suggestingthat the cells are undergoing apoptosis. This observation was furtherconfirmed by studying apoptosis by TUNEL assay .

TABLE 42 Cell Cycle Analysis of F035 Treated Cells Phase of Cell Cycle(Percent of cells) Cell Line F035 (μg/m) SubGo G1 S G2/M MDA-MB-453 01.0 62 26 13 1 1.5 69 21 10 3 1.8 71 16 10 6 2.2 72 19 9.0 MDA-MB-435 01.0 52 35 16 1 1.0 51 36 14 3 13 50 26 12 6 16 50 26 10 MDA-MB-453 &MDA-MB-435 cells were treated with different concentrations of F035 for72 hr at 37° C. Cell cycle analysis was done after propidium iodidestaining as described in the methods.

To understand the mechanism underlying the F035 induced cell kill, theinventors conducted annexin V-FITC binding assay using F035, D1 and G1treated Jurkat cells. Table 43 shows the binding of annexin V to cellstreated with 1 ml of F035, D1 and G1 (15-17%) thereby indicating anapoptotic pathway leading to cell death.

TABLE 43 Jurkat (T-cell leukemia), 72 Hour Cytotoxicity Assay D1Control, D1 Aglycone, D1 w/o Monoterpenes & Monoterpene-Sugar D1 minusD1 minus both Monoterpene- Dose μ * g/ml D1 Control D1 Aglyconemonoterpene monoterpenes sugar 25.000  100 56 7 6 12.500  100 55 4 66.250 62 86 54 3 3 3.125 62 0 43 5 2 1.562 61 0 9 7 1 0.781 61 0 1 4 20.391 57 0 4 4 1 0.195 32 0 1 4 0 0.097 15 0 1 1 0 0.048 0 0 0 0 0.000 00 0 0 IC₅₀ (μg/ml) 0.329 3.634 5.787 >25.000 >25.000

Example 31 Mixture of Triterpene Glycosides Inhibit PI3-Kinase Activity

To study the molecular target(s) of F035, the inventors investigated thePI3-kinase signaling pathway. The results of immunoprecipitation withanti- p85 antibody (adapter protein) probe and subsequent lipid kinaseassay showed that F035 inhibits the activity of PI3-kinase in Jurkatcells. FIG. 45A demonstrates about 50-70% inhibition of PI 3-kinaseactivity with in 2 hr post treatment with F035. By 6 hr 92-95%inhibition of PI3-kinase activity was observed which persisted up to 15hr post treatment. Wortmannin [1 μM, 30 min post treatment], a knownPI3-kinase inhibitor showed similar inhibition of enzyme activity inJurkat cells (FIG. 45A).

Example 32 Mixture of Triterpene Glycosides, D1 & G1, InhibitPhosphorylation of AKT

The inventors determined the effect of F035 and pure extracts on AKT, aserine threonine kinase and a downstream effector of the PI3-kinasesignaling pathway. In contrast to the rapid inhibition of PI3-kinaseactivity, inhibition of AKT phosphorylation did not occur till 15 hrpost treatment . Treatment of Jurkat cells with F035 (2 ml) for 15 hrled to decreased phosphorylation of AKT. However, this treatment alsoled to lowered levels of total AKT protein as can be seen in FIG. 45B.The inventors confirmed the inhibition of AKT activity with puretriterpene glycosides. Pure triterpene glycosides D1 & G1 (2 μg/ml) alsoinhibited AKT phosphorylation and total AKT protein expression. (FIG.45B). Treatment of Jurkat cells with LY 294002 and wortmannin (knownPI3-kinase inhibitors) showed inhibition of AKT phosphorylation.

Example 33 Mixture of Triterpene Glycosides, D1 & G1, Inhibit TNFInduced NF-κB.

In order to further study the mediators of apoptotic pathway, theinventors evaluated the effects of F035, D1 and G1 on the transcriptionfactor NF-κB which has been shown to be involved in apoptosis. Theresults in FIG. 46A show that in Jurkat cells, F035 inhibited theTNF-dependent activation of NF-κB in a dose dependent manner. Untreatedcells and cells treated with F035 alone showed no activation of NF-κB.The inventors also confirmed these results with pure extracts D1 and G1.Pretreatment of cells with 2 ml of G1 and D1 resulted in 54% and 87%decrease in NF-κB levels respectively (FIG. 46B). Cells treated with D1or G1 alone showed no activation of NF-κB (FIG. 46B). Since recentlyPI3-kinase has been shown to regulate NF-κB, pretreatment of cells withwortmannin (1 μM) resulted in almost total inhibition of TNF-inducedNF-κB.

Example 34 Inhibition of iNOS with F035

As the transcription of iNOS is regulated by NF-κB, the inventorsinvestigated the effect of F035 on the induction of iNOS. In U-937 cellswhich were differentiated into macrophages the inventors induced iNOS inresponse to LPS (FIG. 46C). Pretreatment of these cells with F035 (1μg/ml) totally blocked the induction of iNOS. Wortmannin also had asimilar effect on LPS induced iNOS in these cells.

The inventors also examined the effect of F035 on induction of iNOS inJurkat cells. iNOS was induced using PHA and PMA as described in theMethods. The results show that pretreatment of Jurkat cells with F035blocked the induction of iNOS (FIG. 46D).

Example 35 Immunoblot Analysis of PARP Degradation

Apoptosis induced by. F035 & D1 was examined by proteolytic cleavage ofpoly (ADP-ribose) polymerase (PARP). Jurkat cells (2×10⁶/ml) weretreated with F035 (2 μg/ml) and D1 (2 μg/ml) for different lengths oftime. Cell lysates were prepared in buffer containing 20 mM HEPES, 250mMNaCl, 2 mM EDTA, 0.1% NP-40, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 0.5μg/ml benzamidine, 1 mM DTT and 1 mM PMSF. Cellular proteins (60 μg/ml)were separated on a 7.5% SDS polyacrylamide gel and electrotransferredonto a nitrocellulose membrane. The membrane was probed first withmonclonal anti-PARP antibody (PharMingen) and then with anti-mouseantibody conjugated to horse radish peroxidase (HRPO). Protein bandswere detected by chemiluminescense (ECL, Amersham). The extent ofcleavage of the 116-kDa PARP into 85-kDa and 41-kDa peptide products wasused as a measure of apoptosis (Tewari et al., 1995).

Example 36 Assay for Caspase-3 Protease

Caspase-3 activity was measured as described earlier (Enari et al.,1995) with some modifications. Briefly, Jurkat cells (1×10⁶/ml) weretreated with F035, D1 & G1 for different lengths of time. Cytosolicextracts were prepared by repeated freeze thawing in 300 μl ofextraction buffer (12.5 mM Tris, pH 7.0, 1 mM DTT, 0.125 mM EDTA, 5%glycerol, 1 μM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin and 1 μg/mlaprotinin). Cell lystates were diluted 1:2 with ICE buffer (50 mM Tris,pH 7.0, 0.5 mM EDTA, 4 mM DTT and 20% glycerol) and incubated with 20 μMof a caspase 3 substrate (acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin) at37° C. Caspase-3 activity was monitored by the production of fluorescentaminomethylcoumarin, which was measured at excitation 355 nM, emission460 nM using Fluoroscan II (Labsystems, Helsinki, Finland).

Example 37 Detection of Cytochrome C Release from Mitochondria

Release of cytochrome c from mitochondria in response to treatment withF035 was detected by western blotting. Jurkat cells (10×10⁶) weretreated with 2 μg/ml of F035 for 4 and 6 h at 37° C. Cell pellets werewashed in sucrose buffer (0.25M sucrose, 30mM Tris, pH 7.7, 1 mM EDTA).To the cell pellets added 20 μl of sucrose buffer containing 1μM PMSF, 1μg/ml leupeptin, 1 μg/ml pepstatin and 1 μg/ml aprotinin. Cells weredisrupted by douncing 120 times in a 0.3 ml Kontes douncer with a Bpestle (Kontes Glass company). Cellular protein (60 μg) was resolved ona 15% SDS-polyacrylamide gel and electrotransferred onto anitrocellulose membrane. The membrane was probed first with monoclonalanti-cytochrome c antibody (PharMingen) and then with anti-mouseantibody conjugated to horse radish peroxidase (HRPO). Protein bandswere detected by chemiluminescence (ECL, Amersham).

Example 38 F035 and D1 Induce Cleavage of PARP

F035 and D1 induced cleavage of PARP in Jurkat cells in a time dependentmariner. Results in FIG. 47 show that by 4 h both F035 and D1 begin toinduce cleavage of PARP and close to complete cleavage occurs by 6-8 h.This indicates the play of caspases and thereby apoptosis being themechanism involved in the cell kill induced by F035 and D1.

Example 39 Effect of z-vad fmk on F035 Induced Cell Kill

To further confirm the role of caspases in F035 mediated cell kill theinventors studied the effect of z-vad fmk, and inhibitor of caspases oncells treated with F035. Pretreatment of Jurkat cells with 100 μM ofz-vad fmk for 1 h at 37° C. completely reversed the F035 inducedcleavage of PARP (FIG.48).

Example 40 F035 Induces Activation of Caspase 3

The inventors' results so far strongly suggest the role of caspases inF035 induced apoptosis. The inventors next studied the activation ofcaspase 3 in F035, F094, D1 & G1 treated cells since this protease liesimmediately upstream of PARP in the caspase 3 in a time dependentfashion (FIG. 49). Activation starts at 4 h post treatment in all thecases, peaks at 6-8 h and falls thereafter.

Example 41 Cytochrome C Release from Mitochondria by F035

The release of cytochrome c is considered to be the cause of caspase 3activation in some apoptotic pathways. To study if this was true in F035induced apoptosis the inventors, looked at the levels of cytochrome c inthe cytosolic extracts of F035 treated cells. The inventors foundrelease of cytochrome c from the mitochondria of these cells in a timedependent manner (FIG. 50). The inventors see cytochrome c release 4 hfollowing treatment with F035 which coincides with the time whenactivation of caspase 3 and cleavage of PARP begins. Earlier time pointsneed to be studied for cytochrome c release to see if it precedes theactivation of caspase 3.

Example 42 Aeroponic Growth System

In light of the finding that the triterpene compounds of the inventionwere concentrated in the roots and pods of Acacia victoriae plants, itwas desired to create a method for propagating suitable tissue fromwhich the compounds may be isolated. In order to achieve this goal, anaeroponic growth system was designed for the cultivation of Acaciavictoriae roots. The aeroponic system is a closed system in which plantroots are suspended in air and misted with a complete nutrient solution.An 8×4×3.5 ft. box was made out of ¾ inch plywood sheets held togetherwith screws and lined with fiberglass sheets to produce a watertightbox. The top of the box was covered with two (2×8 ft) styrofoam sheets,with 12 circular holes drilled all the way through, although a newdesign incorporating PVC-coated poultry wire covered with opaqueco-extruded white-on-black polyethylene is being considered as a chambercover for future work. A program-repeating timer was used to mist theroots for a period of 12 seconds every 4.5 minutes.

The plumbing system design for the aeroponic chamber is a closed systemconstructed of ¾ inch PVC with six whirl-jet hollow cone polypropylenespray nozzles. A reservoir of 720 liters of nutrient solution ismaintained in the bottom of the chamber, and sprayed on the roots of theplants from below using an external pump. The pump used was a LittleGiant 4-MD 3250 RPM, 1/12 hp pump.

The pump is controlled by a Tork repeating timer set for intervals of 30seconds of spray every 4.5 minutes. Temperatures were monitored with aTaylor electronic indoor/outdoor minimum/maximum thermometer andrecorded by hand. Two Visi-therm 300W submersible aquarium heaters wereused to heat the nutrient solution during the winter months, which wassufficient to keep the plants actively growing without heating thesurrounding air in an unheated and uncooled outdoor shade-house inTucson, Ariz.

The nutrient solution contained all of the essential elements the plantsneeds to complete its life cycle. Despite the fact that different plantsrequire different levels and formulations for optimum growth, anover-all, single-balanced solution gives satisfactory results. Thecomposition of the solution is given below, in Table 44.

TABLE 44 Aeroponic Nutrient Solution Compound Element Concentration(ppm) Calcium Nitrate N 150 Potassium nitrate Ca 146 Potassium Nitrate K200 Mono-potassium phosphate P 90 Magnesium Sulphate Mg 50 S 134 10%Fe-chelate Fe 5 Copper Sulfate Cu 0.07 Manganese Chloride Mn 0.8 SodiumMolybdate Mo 0.03 Boric Acid B 0.3 Zinc Sulfate Zn 0.1

Seeds of Acacia victoriae were then scarified and sown in a soil-lessmix composed of 50% peat moss and 50% vermiculite. The seedlings werewatered twice a day and fertilized with a single dose of osmacote. Oncethe seedlings were between 15-20 cm long, which was usually achievedafter 3-4 months of growth, the root balls were washed thoroughly toremove all traces of peat moss and vermiculite. Next the roots wereslipped through holes in the Styrofoam boards, and the top of theseedlings was supported from above by twine coming down from thegreenhouse structure. A 7.0 cm tubular piece of foam was wrapped aroundthe crown of the seedlings to prevent misting of the leaves and thesurrounding area. The box was then filled with approximately 30 cm ofnutrient solution, and the pump turned on.

Once the seedlings were in position and being misted, maintenance waslimited to training the growing seedlings up the twine using plasticclips and replenishing the nutrient solution as the level dropped below10 cm. While the seedlings were growing inside a greenhouse, temperaturecontrol of the nutrient solution was not necessary. However, if theaeroponic box is subjected to ambient environmental conditions, it isrecommended to increase the nutrient solution temperature to 70° F. sothat the plants will not become dormant during winter months.

For harvesting of roots, the root mass of a single plant is rinsed withwater directly in the aeroponic box and the root mass is cut withscissors a few inches above the sprayer. The excess water is removed bypatting dry with paper towels, followed by weighing of the sample. Theroot mass is then cut in 3-4 inch sections with scissors and subject tochemical extraction, as described above. Alternatively, for continualharvest of roots, the pump is turned off and roots are clipped from thegrowing root mass. These roots are then cut into 3-4 inch sections andextracted as described. Care is taken not to damage the non-harvestedroots.

A number of advantages were realized by growing plants in the aeroponicsystem. First, the growth of the plants was approximately twice thatachieved with conventional growing techniques. Second, the roots can beeasily harvested as needed without harming the plants. This cutting ofroots further leads to extensive lateral growth of fibrous roots.Therefore, the roots could be harvested several times a year. Further,the aeroponically grown plants flowered in their first year of growth,compared to 3-5 years for plants grown outdoors.

Example 43 Tissue Culturing and Germination of Acacia victoriae

Seeds/Substrate: Seeds were harvested from plants growing at the CampusAgricultural Center, University of Arizona, Tucson, Ariz. Seeds werewashed thoroughly in tap water with an anti-microbial soap (Vionex, ViroResearch International Inc., USA Durango, Colo.), then treated withcommercial bleach 20% (v/v) for 15 min. After repeated washing indeionized water, they were treated with boiling water (ca 200 ml for 100seeds) and left to cool overnight. Then they were treated with 20%(v/v)commercial bleach for 20 min, rinsed 2-3 times in sterile deionizedwater, and cultured on MS (Murashige and Skoog, 1962) and ½ strength MSmedium. The medium was supplemented with MS vitamins, 2%(w/v) sucroseand gelled with either 0.7% agar or 0.2% Gelrite. In one study, theseeds were scarified with concentrated sulfuric acid, rinsed in sterilewater, and cultured on medium. All media was autoclaved at 121° C. for15 min. Cultures were maintained at 25+2° C. under 16-h lightphotoperiod at 1000 lux produced from cool white fluorescent tubes. Eachstudy contained 18 replications.

Propagation: Shoot tips and nodal segments excised from three-week-oldseedlings were cultured on MS medium alone and also MS supplemented with0.1 mg/L of auxins (IAA, NAA or IBA) and BAP (0.1, 0.3, 0.5, 1.0 and 1.3mg/L) either separately or in combinations. For rooting of shoots IAA(0.1 mg/L), IBA (0.1 and 0.6 mg/L) and NAA (0.1 and 0.2 mg/L) weretested. For transfer to soil, plantlets were removed from culture tubes,the roots were washed with tap water to remove the nutrients adhering toroots and the transferred to pots filled with desert-type soil. Theplants were covered with Magenta boxes to maintain humidity and keptunder mist and low light for 3 wk. After 3 wk, the Magenta boxes wereremoved and the plants were transferred from the mist to higher light inthe greenhouse.

Induction of callus: Callus tissue was induced from hypocotyl and rootsegments excised from 3-week-old in vitro germinated seedlings. Theexplants were cultured on MS medium supplemented with 2,4-D (1 mg/L),NAA (0.5 & 1 mg/L), IAA (0.2 and 1 mg/L), Thidiazuron (0.2 mg/L),Dicamba (0.2 & 2 mg/L), BAP (0.3 mg/L) and KN (0.5 and 3 mg/L) eitherindividually or in combinations.

Seed Germination: Seeds treated with hot water germinated with theemergence of the radicle in 3-4 days and the complete plantlets wereobtained within 1 wk. Seeds cultured without hot water treatment did notgerminate. A high percentage of seeds germinated on medium solidifiedwith Gelrite (0.2%) as compared to agar (0.7%). The maximum germinationpercentage of 88.7% was noted on half strength MS medium solidified withGelrite. The germination responses on different media are summarized inTable 45.

TABLE 45 Seed Germination of Acacia victoriae No. of Media No. of SeedsCultured Seeds^(a) Germinated MS (agar solidified) 42   36 (85.7) MS(agar solidified) 41 24 (58) (decoated with sulfuric acid) ½ strength MS(agar 60 48 (80) solidified) ½ strength MS (Gelrite 133  118 (88.7)solidified) ^(a)Numbers in parentheses are percent germination.

Transplantable seedlings were obtained in 3-4 wk time. The seeds of A.victoriae have low germination rates in vivo due to high levels of seeddormancy (Kaul and Ganguly, 1965; Grice and Westoby, 1987). To overcomedormancy, seed coats must be either nicked with a sharp instrument,subjected to acid scarification, or covered with boiling water and leftto cool in the water overnight. The inventors found that the germinationpercentage can be increased up to 88.7% by using the boiling watertreatment and subsequently culturing the seeds on ½ MS medium gelledwith 0.2% Gelrite. According to Larsen (1964), A. victoriae seeds underin vivo conditions treated with boiling water can increase germinationby 36%. Without pretreatment, the germination percentage was 19.4% (Kauland Ganguly, 1965). In addition, it took 12 days for the radicle toemerge and complete seedlings were recovered after 79 days. In ourprotocol, the percent germination is increased (88.7%) andtransplantable seedlings could be obtained in 3-4 wk time.

Shoot tip cultures: To investigate shoot multiplication, the shoot tips(about 1.0 cm in length) were cultured on either MS alone or MSsupplemented with BA, and BA in combination with IAA. On MS alone theshoots had poor vigor, and a poor root growth (1-3 roots /culture). Onmedium containing BA(1.3 mg/L), the shoot tips produced multiple shoots(average of 3.94 shoots/culture). Among the multiple shoots, one or twoshoots elongated and attained a height of 8.6 cm in 4 wk. Thecombinations of BA and IAA also favored multiple shoot induction. Theresponses are summarized in Table 46.

TABLE 46 Effect of Different Levels of BA And IAA (0.2 Mg/L) on MultipleShoot Induction in Acacia victoriae. Media* Average No. of shoots ShootLength BA (mg/L IAA (mg/L per shoot tip (cm) 1.3 0 3.94 + 1.846   8.6 +3.0258 0.1 0.2 1.6 + 0.599  6.8 + 3.002 0.3 0.2 1.9 + 0.7071 5.8 + 2.7940.5 0.2 2.8 + 1.1659 5.1 + 2.501 1.0 0.2 4.9 + 2.075  3.2 + 1.468 *MS.Data represents an average of 18 replicates + SE.

At higher BA concentrations (1.0 & 1.3 mg/L), the number of shootsincreased. The combination of BA (1 mg/L)+IAA (0.2 mg/L) was found to bebetter for shoot multiplication. Callus was observed at the cut ends inall the BA-IAA combinations. Kaur, et al. (1998), reported thesynergistic effect of BA-NAA on shoot bud induction in Acacia catechuand higher levels of NAA (1-2 mg/L) were not beneficial. They alsostated that IAA was not effective in enhancing shoot bud formation; butinstead callus was produced from the base of the explants.

To investigate rooting, in vitro-developed shoots were excised andtransferred to medium containing IAA, NAA or IBA. The responses aresummarized in Table 47. Among the treatments tested, ½ MS+NAA (0.2mg/L)was found better for rooting. Almost 100% of the shoots rooted. Theshoots attained a height of 9-11 cm in four wk. In Acacia catechu (Kaur,et al., 1998) reported that intermittent callus formation at thejunction of root and shoot and they employed reduced sucrose level from3% to 1.5% to control the callus. Similar results were also reported inFeronia limonia (Purohit and Tak, 1992) and Acacia auriculiformis (Das,et al., 1993). In the present investigation, slight callusing was alsonoted at 3% sucrose and it was minimized at 2% sucrose. The rootedshoots were transferred to the greenhouse. The survival aftertransferring was 100%. The plantlets were acclimatized under mist for 3wk and later the plantlets were grown in the regular greenhouse.

TABLE 47 Effect of IAA, NA and IBA on Rooting of Shoots of Acaciavictoriae No. of shoots No. of shoots^(a) Mean No. of Media culturedrooted roots/culture MS 14   6 (42.8)   2 + 0.816 MS + IAA (0.1) 12   8(66.6)  3.6 + 1.316 MS + IBA (0.1) 10  6 (60)   3 + 0.816 MS + IBA (0.6)14  8 (57)  1.6 + 1.111 MS + NAA (0.1) 10  6 (60) 2.16 + 1.067 ½ MS +NAA (0.2) 14  14 (100) 3.07 + 1.032 ^(a)Numbers in parentheses arepercent rooting.

Nodal segment cultures: Nodal segments (cotyledonary node) excised fromin vitro germinated seedlings were cultured on MS medium supplementedwith 0.1 mg/L IAA, NAA or IBA. Only one or two axillary shoots developedper explant. However, the growth of these shoots was slow. Hence, nodalexplants were not used for further studies.

Induction of callus from hypocotyl and root segments: Callus was inducedfrom hypocotyl segments excised from 3-wk-old in vitro germinatedseedlings. The callus developed on 2,4-D (1 mg/L), Thidiazuron (0.2mg/L), Dicamba (0.2 mg/L) was greenish, compact and hard. The quantityof callus developed was moderate in most of the concentrations tried(Table 51). Profuse callus development was noted on MS mediumsupplemented with 2,4-D (4 mg/L)+IAA (1 mg/L)+NAA (1 mg/L).

Root segments excised from three-week-old in vitro germinated seedlingswere cultured on MS medium supplemented with 2, 4-D (1 mg/L) alone and2,4-D in combination with KN (0.5 mg/L) showed the development of lightyellowish soft callus with a few roots developing from the callus. Thecallusing was noted in 100% of the cultures. Whitish, soft, friable andprofuse callusing was noted from root segments on medium added with BA(0.3 mg/L)+IAA (0.2 mg/L). Light yellowish profuse callusing was notedon the root segments cultured on medium added with 2,4-D (4 mg/L) incombination with 1 mg/L each of IAA and NAA. A similar type of callusingwas noted in Thidiazuron (0.2 mg/L)+Dicamba (2 mg/L) and IAA (0.1 mg/L).Root segments cultured on medium with Dicamba (2 mg/L)+IAA (0.1 mg/L)formed light green compact hard callus. Attempts to regenerate theplantlets from the callus were not successful. Variation among explanttypes with respect to callus induction has been reported in severalwoody species such as Albiizzia lebbeck (Lakshmana Rao and De, 1987) andLonicera japonica (Georges, et al., 1993). In the inventors' studies,they also found that there is a difference between hypocotyl- androot-derived callus developed on the identical medium. Calli developedfrom hypocotyl on BA-IAA combinations were light greenish, hard andcompact, whereas from the root segments it was whitish, soft, friableand also showed root differentiation from the callus in some of thecombinations. In Dalbergia latifolia the callus on regenerating mediabecame compact, hard and dark green and shoot buds were differentiated(Pradhan, et al., 1998). In the inventors' studies, a similar type ofcallus development was noted, but such callus failed to regenerate. Inthis investigation the inventors showed that A. victoriae can bepropagated in vitro from shoot tips. The technique standardized isuseful for the micropropagation of elite individuals detected among theheterogeneous seedling populations and maintenance of elite lines forfuture studies.

TABLE 48 Development of Callus from Hypocotyl and Root Segments ofAcacia victoriae Nature of callus Media* Hypocotyl Root 1. MS + 2,4-D(1) Moderate, green Moderate, yellow 2. MS + TD (0.2) Scanty Scanty 3.MS + Dicamba (2) Moderate, compact Moderate, soft green yellow 4. MS +2,4-D (1) + KN (0.5) Scanty, green Scanty, light green 5. MS + KN (3) +NAA (0.5) Moderate, white Scanty, light green 6. MS + TD (0.2) +Moderate, light Moderate, soft green yellow 7. MS + Dicamba (2) +Scanty, compact Scanty, light IAA (0.2) yellow green 8. MS + 2,4-D (4) +Profuse, green, Moderate, yellow IAA (1) + NAA (1) compact, hard soft 9.MS + BA (0.3) + IAA (0.2) Moderate, compact Profuse, white *Numbers inparentheses are mg/L.

Example 44 Induction of Hairy Roots from Acacia victoriae for theProduction of Anti-Cancer Compounds

Infection of Acacia victoriae plant material with Agrobacteriumrhizogenes leads to the integration and expression of T-DNA in the plantgenome, which causes development of a hairy root phenotype (Grant etal., 1991). Hairy root cultures grow rapidly, show plagiotropic rootgrowth and are highly branched on hormone-free medium. Hairy roots alsoexhibit a high degree of genetic stability (Aird et al., 1988). Manydicotyledonous plants are susceptible to A. rhizogenes, and plants havebeen regenerated from hairy root cultures in many species (Christey,1997).

Genetic transformation and the induction of hairy roots were performedby the inventors as a method for the production of the activetriterpenes from A. victoriae. The natural ability of the soil bacteriumAgrobacterium rhizogenes to transform genes into a host plant genomeresults in roots being formed at the site of infection is used toproduce hairy root cultures. Hairy roots are characterized by numerousfast growing, highly branched adventitious roots, which continues togrow in vitro on hormone-free medium.

The inventors demonstrated induction of hairy roots in Acacia victoriaeusing Agrobacterium rhizogenes strain R 1000 (an engineered strain ofAgrobacterium tumefaciens strain to which Agrobacterium rhizogenesplasmid pRiA₄ has been inserted, ATCC Number 43056). The production ofthe compound of interest in hairy roots was confirmed by HPLC. Inductionof hairy roots was carried out as follows. First, Acacia victoriae seedswere collected from field-grown plants in Tucson, Ariz. Boiling waterwas poured over the seeds, which were soaked overnight as the watercooled and surface sterilized in 15% commercial bleach for 30 minutes.After repeated washing in sterile water, seeds were cultured on liquidMS medium (Murashige and Skoog, 1962) supplemented with MS vitamins and2% sucrose in 250 ml conical flasks with 50 ml medium. The cultures weremaintained in a gyratory shaker in a growth room at 25±2 ° C. in thedark. After four days of culture, embryo-axis were excised from thegerminating seedlings and used for agroinfection.

Prior to agroinfection, Agrobacterium rhizogenes strain R1000 was grownovernight on YENB medium. YENB medium was prepared by adding 7.5 g/LYeast Extract and 8 g/L Nutrient Broth (Difco Laboratories, Detroit,MI). The embryo-axis of the explants was infected with a fine stainlesssteel needle that had been dipped in bacterial solution. Afterinfection, a drop of bacterial suspension (1:20 with MS medium) was puton the surface of the explants. Then the explants were transferred to MSmedium and MS medium with acetosyringone (100 μM) (3,5 dimethoxy-4hydroxy-acetophenone, Aldrich Chem. Co, Milwaukee, Wis.) forco-cultivation. Co-cultivation was carried out for three days in thedark. After three days of co-cultivation, the explants were transferredto MS+Cefotaxime (500 mg/l, Agri-Bio, North Miami, Fla.) to control thebacterial overgrowth. Root initiation was noted at the site of infectionmostly from the young developing leaves from the embryo-axis in 3-4weeks time. After 4 weeks, the explants along with the roots weretransferred to MS medium alone and the dark incubation was continued forthe development of hairy roots. Hairy root development was noted after afurther 8 weeks. The hairy roots thus developed were multipliedroutinely on MS medium by subculturing. The transgenic nature of thehairy roots was confirmed by PCR™ using a set of primers to amplify aportion of the rol B gene. The primers used were as follows:

1) 5′ GAGGGGATCCGATTTGCTTTTG 3′ (SEQ ID NO. 7) 2)5′ CTGATCAGGCCCCGAGAGTC 3′ (SEQ ID NO. 8)

A 50 μl PCR™ reaction mix contained the primers (1 μM finalconcentration each), Taq polymerase (1.0U), 125 μM each dNTP, 1×PCR™reaction buffer, 1.5 mM Mg Cl₂, 300 ng of isolated DNA. PCR™ conditionsemployed were 92° C. initial denaturation for five min followed by 35cycles of 92° C. 50 seconds, 55° C. 1 min for annealing, 72° C. 1 and ½min for extension and 72° C. 7 min final extension. A 645 by fragmentwas amplified.

Hairy root cultures in liquid medium: To optimize the conditions for thegrowth, hairy roots growing on MS semi-solid medium were excised andcultured in MS liquid medium in different capacity flasks (125, 250, 500and 1,000 mL) with 20, 50, 100 and 400 mL medium respectively. Theinitial hairy root innoculum was 6 gm/L. The growth of hairy roots wasalso tested in the following basal media: MS, Nitsch and Nitsch (N andN) (1969), Schenk and Hilderbrandt (SH) (1972) and Woody Plant Medium(WPM) (Lloyd and McCown, 1981). To test the effect of different carbonsources on hairy root growth, 2% (w/v) of each of the following wasadded to MS medium: sucrose, glucose, fructose and mannose. The effectof gibberellic acid (0.1, 0.5 and 1 mg/L) on hairy root growth wastested by adding the filter-sterilized solution to MS medium afterautoclaving.

Initiation of roots at the site of infection was noted in 3-4 weeks.Four independently transformed hairy root clones were established fromembryo axes infected with R1000 strain in the presence of acetosyringone(100pM). The embryo axes co-cultivated with A. rhizogenes withoutacetosyringone did not develop hairy roots (Table 54). Three daysco-cultivation in the presence of acetosyringone was found optimum forinduction of hairy roots. A promoting effect of acetosyringone has beenreported in Salvia militiorrhiza (Hu and Alfermann, 1993). The resultsshowed that acetosyringone, an activator of the vir genes ofAgrobacterium, increased the transformation frequency. Similarly, inthis study, acetosyringone was required to induce hairy roots.

The transformed nature of the roots was confirmed by PCR™ amplificationusing a set of primers to amplify a portion of rol B gene. A diagnosticfragment of 645 by was amplified in the four hairy root clones tested.

The hairy roots grown on liquid medium developed vigorously. Among thedifferent basal media tested, MS medium was found best for hairy rootgrowth (Table 56). In a 125 mL flask, there was a 268-fold increase ingrowth in 4 weeks. With WPM and N and N medium, there was a 254- and196-fold increase respectively. B₅ and SH medium did not favor theoptimal growth of hairy roots. Hairy roots slowly started browning onthese two media. In one study, hairy roots were grown in differentcapacity flasks (125, 250, 500 and 1000 mL) with 20, 50, 100 and 400 mLMS medium, respectively. The growth kinetics are summarized in Table 55.Initially, the growth of hairy roots is vigorous and attained a25.77-fold increase in 4 weeks in 125 mL flasks with a starting inoculumof 150 mg. As the flask capacity was increased, the growth of rootsslightly decreased.

The growth of hairy roots can be sensitive to medium composition,especially mineral ions and carbon source (Wysokinska and Chmiel, 1997).For Acacia victoriae, five different basal media (MS, N and N, SH, WPMand B₅) were tested for effect on hairy root growth. MS medium was foundbest for growth. Sasaki et al. (1998) compared the growths of Coleusforskohlii hairy roots on various nutrient media and found that WPM wasbest for hairy root growth.

In this study, sucrose favored the growth of hairy roots compared toother carbon sources (fructose, glucose and mannose). The maximum growth(24.52-fold increase) was found in sucrose-containing medium.Glucose-containing medium was slightly inhibitory for growth, andmannose completely inhibited the growth (Table 57). In Catharanthusroseus, catharanthine production could be doubled by the use of fructoseas a carbon source in the medium. However, the authors reported that useof fructose resulted in an approximately 40% decrease in growth comparedto sucrose (Jung et al., 1992).

Hairy roots do not require the addition of exogenous growth regulatorsfor continued growth because genes that increase sensitivity to auxinare present in the Ri plasmid (Wysokinska and Chmiel, 1997). However,reports are available wherein exogenous hormones stimulate growth. Theinventors tested the effect of gibberellic acid (0.1,0.5 and 1.0 mg/L)on hairy root growth. The growth of hairy roots was best in mediumwithout GA₃, as compared to GA₃-containing medium (15.77-fold increase).Different levels of GA₃ did not affect the growth significantly (Table58). In Artemisia, GA₃ did not enhance the overall biomass accumulation,but it facilitated reaching stationery phase sooner than cultures grownon medium without GA₃ (Smith et al., 1997). Rhodes et al., (1994) foundthat the response of hairy roots of Brassica candida to GA₃ dependedlargely on the clone examined. However, they observed that generally GA₃exerted a positive effect on growth and a reduction in the accumulationof alkaloids accompanied with changes in patterns of production. Ohkawaet al., (1989) reported GA₃ at concentrations of 10 ng/L and 1 mg/Laccelerated growth, enhanced elongation, and increased lateral branchingin Datura innoxia hairy roots. Zobel (1989) suggested that GA₃ acts as aCO₂ analog for root growth. For Acacia victoriae hairy roots, GA₃ didnot enhance the growth, which might indicate a differential response forvarious genotypes.

The use of hairy root cultures of Acacia victoriae will provide asuitable means for uniform culture of plant tissue from which thetriterpene glycoside compositions of this invention, which includeisolated mixtures or individual purified compounds, can be isolated.

TABLE 49 Agrobacterium rhizogenes Strain R1000 Infection of Embryo Axesof Acacia victoriae for Hairy Root Production No. of No. embryoexplants^(a) No. of roots *Media for axis with root with hairy rootTreatment co-cultivation infected development morphology Control MS 20 —— (non infected) MS + Aceto. 21 — — Infected MS 33 5 (15) MS + Aceto. 389 (23) 4 (17.3) *Acetosyringone (100 μM) was added after autoclavinginto MS medium for co-cultivation ^(a)Number in parenthesis indicatespercentage.

TABLE 50 Effect of Different Flask Sizes on the Growth of Hairy Roots ofAcacia victoriae Initial Fresh Fresh Weight Flask size weight after 4weeks (mL) (mg) (mg)^(a) Fold increase 125 150 3866 ± 0.569 25.77 250300 6903 ± 0.344 23.01 500 1200 11817 ± 0.998  9.84 1000 2400 40080 ±3.479  16.70 ^(a)Data represents an average of 6 replicates ± S.E., 125,250, 500 and 1000 mL capacity flasks with 25, 50, 100 and 400 mL MSmedium.

TABLE 51 Effect of Different Basal Media and Flask Size an the Growth ofHairy Roots of Acacia victoriae Initial Fresh. Fresh. Weight FlaskSize^(b) Weight after 4 weeks Media^(a) (mL) (mg) weeks(mg) FoldIncrease MS 125 10 2681 268 B₅ 125 10 1933 193 N and N 125 10 196 196 SH125 10 170 170 WPM 125 10 2549 254 MS 250 300 751 25 B₅ 250 300 57 19 Nand N 250 300 591 19.7 SH 250 300 54 18 WPM 250 300 659 21 ^(a)MS =Murashige and Skoog; B₅ = Gamborg's; N and N = Nitsch and Nitsch; SH =Schenk and Hilderbrandt; WPM = Woody plant medium. ^(b)125 and 250 mLflasks with 25 and 50 mL medium.

TABLE 52 Effect of Various Carbon Sources in MS Medium on the Growth ofHairy Roots of Acacia victoriae Carbon source^(a) Fresh weight after (2%W/V) 4 weeks (gm)^(b) Fold increase Sucrose   7.356 ± 0.543^(c) 24.52Glucose 2.87 ± 0.53 9.56 Fructose 5.85 ± 1.55 19.5 Mannose 0.305 ± 0.0651.01 ^(a)2% (w/v) ^(b) The initial F.W. for each treatment was 300 mg.^(c)Data represents an average of 6 replicates ± S.E.

TABLE 53 Effect of GA₃ on the Growth of Hairy Roots of Acacia victoriaeFresh weight after^(a) GA₃ 4 weeks (mg/L) (gm) Fold increase 0  6.512 ±1.569^(b) 21.70 0.1 4.732 ± 0.086 15.77 0.5 4.634 ± 0.088 15.44 1 4.310± 0.344 15.44 ^(a)The initial F.W. for each treatment was 300 mg.^(b)Data represents an average of 6 replicates ± S.E.

Different media were tested for growth of hairy roots. Best growth wasobtained on MS medium containing 2% sucrose. The effect of differentcapacity flasks and gibberellic acid was tested on the growth of hairyroots. The hairy roots were also grown on MS liquid medium on gyratoryshaker in a 125 ml conical flask with 20 ml medium. An increase ingrowth of 84 fold was noted in 4 weeks. The production of triterpenesaponins corresponding to those identified in F035 was confirmed by HPLCanalysis with a standard authentic sample.

All of the composition and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved.

All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the claims.

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The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A mixture comprising one or more isolated triterpene glycosidescharacterized by the following properties: a) isolatable from the tissueof Acacia victoriae; b) containing at least one triterpene glycosidehaving a molecular weight of from about 1800 to about 2600; c) theability to induce cytotoxicity in a Jurkat cell; and d) the ability toinduce apoptosis in a Jurkat cell.
 2. The mixture of claim 1, whereinsaid mixture induces cytotoxicity in said Jurkat cell with an IC₅₀ offrom about 0.12 μg/ml to about 0.40 μg/ml.
 3. The mixture of claim 1,wherein said apoptosis is induced when administered to said Jurkat cellat a concentration of from about 100 ng/ml to about 400 ng/ml.
 4. Themixture of claim 3, wherein said apoptosis is induced when administeredto said Jurkat cell at a concentration of from about 200 ng/ml to about400 ng/ml. 5-119. (canceled)
 120. The mixture of claim 1, wherein themixture further comprises a pharmacologically acceptable medium. 121.The mixture of claim 120, wherein the pharmaceutically acceptable mediumis a buffer, a solvent, a diluent, an inert carrier, an oil, or anedible material.
 122. The mixture of claim 1, wherein the mixture isfurther defined as an ointment, a crème, or a tablet.
 123. The mixtureof claim 1, wherein the mixture comprises dried or ground Acaciavictoriae pod.
 124. The mixture of claim 1, wherein the mixture iscomprised in a capsule.
 125. The mixture of claim 1, wherein the mixturecomprises a compound of molecular formula:

wherein (a) R₁ is selected from the group consisting of hydrogen, C₁-0₅alkyl, and an oligosaccharide; (b) R₂ is selected from the groupconsisting of hydrogen, C₁-C₅ alkyl, and an oligosaccharide; (c) R₃ isselected from the group consisting of hydrogen, hydroxyl, C₁-C₅ alkyl,C₁-C₅ alkylene, C₁-C₅ alkyl carbonyl, a sugar, and a monoterpene group;and (d) the formula further comprises R₄, wherein R₄ is selected fromthe group consisting of hydrogen, hydroxyl, C₁-C₅ alkyl, C₁-C₅ alkylene,C₁-C₅ alkyl carbonyl, a sugar, C₁-C₅ alkyl ester, and a monoterpenegroup, wherein R₄ may be attached to the triterpene moiety or themonoterpene moiety.
 126. The mixture of claim 125, wherein R₁ is anoligosaccharide.
 127. The mixture of claim 126, wherein theoligosaccharide comprises a fucose, a xylose, or an N-acetyl glucosamineresidue.